MedWorm.com provides a medical RSS filtering service. Over 6000 RSS medical sources are combined and output via different filters. This feed contains the latest items from the 'Journal of Immunological Methods' source. Wed, 08 Feb 2012 06:42:57 +0100 http://www.medworm.com/index.php?rid=5660374&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D22301270%26dopt%3DAbstract Authors: Selman L, Henriksen ML, Brandt J, Palarasah Y, Waters A, Beales PL, Holmskov U, Jørgensen TJ, Nielsen C, Skjodt K, Hansen S Abstract Collectin 11 (CL-11), also referred to as collectin kidney 1 (CL-K1), is a pattern recognition molecule that belongs to the collectin group of proteins involved in innate immunity. It interacts with glycoconjugates on pathogen surfaces and has been found in complex with mannose-binding lectin-associated serine protease 1 (MASP-1) and/or MASP-3 in circulation. Mutation in the CL-11 gene was recently associated with the developmental syndrome 3MC. In the present study, we established and thoroughly validated a sandwich enzyme-linked immunosorbent assay (ELISA) based on two different monoclonal antibodies. The assay is highly sensitive, specifi... Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=5660374 Tue, 31 Jan 2012 05:00:00 +0100 5660374 http://www.medworm.com/index.php?rid=5660375&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D22293629%26dopt%3DAbstract Authors: Whitelegg AM, Birtwistle J, Richter A, Campbell JP, Turner JE, Ahmed TM, Giles LJ, Fellows M, Plant T, Ferraro AJ, Cobbold M, Drayson MT, Maclennan CA Abstract The measurement of antibody responses to vaccination is useful in the assessment of immune status in suspected immune deficiency. Previous reliance on enzyme-linked immunoabsorbent assays (ELISA) has been cumbersome, time-consuming and expensive. The availability of flow cytometry systems has led to the development of multiplexed assays enabling simultaneous measurement of antibodies to several antigens. We optimized a flow cytometric bead-based assay to measure IgG and IgM concentrations in serum to 19 antigens contained in groups of bacterial subunit vaccines: pneumococcal vaccines, meningococcal vaccines, Haemoph... Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=5660375 Tue, 24 Jan 2012 05:00:00 +0100 5660375 http://www.medworm.com/index.php?rid=5642812&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D22269771%26dopt%3DAbstract Authors: Dong J, Hasan S, Fujioka Y, Ueda H Abstract Open sandwich immunoassay (OS-IA) utilizes antigen-dependent stabilization of antibody variable region to quantify various antigens, enabling noncompetitive detection of small molecules with a broad working range. To further improve its detection sensitivity, here we employed phage-based immuno-PCR approach, wherein OS-IA and quantitative PCR methodologies were combined with the use of immobilized V(L) fusion protein and filamentous phages displaying V(H) fragment, whose DNA was extracted for PCR amplification. This approach significantly enhanced the assay sensitivity for small molecule antigens osteocalcin (BGP) peptide and 17beta-estradiol. PMID: 22269771 [PubMed - as supplied by publisher] (Source: Journal of Immunologica... Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=5642812 Tue, 17 Jan 2012 05:00:00 +0100 5642812 http://www.medworm.com/index.php?rid=5642811&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D22269772%26dopt%3DAbstract Authors: Van Nuffel AM, Tuyaerts S, Benteyn D, Wilgenhof S, Corthals J, Heirman C, Neyns B, Thielemans K, Bonehill A Abstract The efficacy of cancer immunotherapy can be improved by treatment with full-length tumor antigen and by combining several antigens. This approach allows the induction of a broad immune response irrespective of the patient's HLA type which at the same time challenges immune monitoring. Also, the number of available lymphocytes is most often limited and minimal in vitro restimulations of the lymphocytes should maintain information about the actual in vivo situation. To overcome these hurdles, we developed a method to measure the CD8(+) and CD4(+) T-cell responses directly ex vivo. Skin biopsies taken from dendritic cell (DC)-induced DTH reactions from melanoma... Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=5642811 Mon, 16 Jan 2012 05:00:00 +0100 5642811 http://www.medworm.com/index.php?rid=5642810&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D22273672%26dopt%3DAbstract In this study, DEC205 knockout (KO) mice were immunized with this full-length extracellular domain protein of hDEC205. One of the 3 immunized DEC205 KO mice was chosen for the highest anti-hDEC205 titer by flow cytometric analysis of serum samples on CHO cells stably expressing hDEC205 (CHO/hDEC205 cells) and used for hybridoma fusion. From a single fusion, more than 400 anti-hDEC205 hybridomas were identified by flow cytometric screen with CHO/hDEC205 cells, and a total of 115 hybridomas secreting strong anti-hDEC205 mAb were saved and named HD1 through HD115. To characterize in detail, 10 HD mAbs were chosen for superior anti-hDEC205 reactivity and further subjected to cloning and purification. Interestingly, out of those 10 chosen anti-hDEC205 HD mAbs, 5 mAbs were also strongly reactive... Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=5642810 Mon, 16 Jan 2012 05:00:00 +0100 5642810 http://www.medworm.com/index.php?rid=5624713&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D22265885%26dopt%3DAbstract Authors: Chan LL, Zhong X, Pirani A, Lin B Abstract Cell proliferation is an important assay for pharmaceutical and biomedical research to test the effects of a variety of treatments on cultured primary cells or cell lines. For immunological studies, the ability to perform rapid cell proliferation analysis allows the identification of potential biological reagents for inducing or inhibiting immune cell proliferation. Current cell proliferation analysis methods employ flow cytometry for fluorescence detection of CFSE-labeled cells. However, conventional flow cytometers require a considerable amount of cells per sample, which becomes an issue for kinetic measurements with rare cell population due to the lack of samples for flow cytometric analyses at multiple time points during proli... Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=5624713 Sun, 15 Jan 2012 05:00:00 +0100 5624713 http://www.medworm.com/index.php?rid=5624712&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D22265970%26dopt%3DAbstract Authors: Ebert LM, Macraild SE, Davis ID, Cebon J, Chen W Abstract Antigenic epitopes recognized by FoxP3(+) regulatory T cells (Treg) are poorly defined, largely due to a lack of assays for determining Treg specificity. We have developed a novel approach for detecting human Treg specific to peptide antigen, utilizing down-regulation of surface CD3 as a read-out of antigen recognition. Culture conditions and re-stimulation time have been optimized, allowing the detection of even very rare Treg, such as those specific to tumor antigens. PMID: 22265970 [PubMed - as supplied by publisher] (Source: Journal of Immunological Methods) Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=5624712 Fri, 13 Jan 2012 05:00:00 +0100 5624712 http://www.medworm.com/index.php?rid=5624714&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D22261191%26dopt%3DAbstract Authors: Otani N, Yamanishi K, Sakaguchi Y, Imai Y, Shima M, Okuno T Abstract Though cell-mediated immunity (CMI) against varicella-zoster virus (VZV) is critical for prevention of the onset of herpes zoster (HZ), clinicians currently lack a simplified procedure to monitor CMI. We have recently developed an assay, called the IFN-γ release assay, and showed that it is a simple and reliable method to determine VZV-specific CMI. In the present study, we applied an IR assay to measure the VZV-specific CMI of patients with HZ. VZV-specific CMI levels were significantly high at the onset of the disease, but were decreased several weeks later. In contrast, CMI VZV-specific antibody titers increased in convalescent phase compared to those in acute phase. Thus, this technology is likely to... Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=5624714 Thu, 12 Jan 2012 05:00:00 +0100 5624714 http://www.medworm.com/index.php?rid=5624716&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D22251662%26dopt%3DAbstract Authors: Li Pira G, Ivaldi F, Manca F Abstract Adherent antigen presenting cells (APC) pulsed with protein or peptide antigens were used to capture specific CD4 or CD8 T-cells derived from established T-cell lines or from PBMC of immune subjects based on physiological interaction between TCR and MHC-peptide complex. This method could be applied independently of epitope specificity, HLA restriction alleles, activation markers and secreted cytokines, parameters required by other methods for selection of specific T cells. Non specific T-cells were removed by applying a 1g force that did not affect binding of specific T-lymphocytes. Lymphocyte selection was specific and the average recovery was 36% for CD4 T-cells. CD8 T-cells proved trickier to purify, since solid phase APC were recog... Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=5624716 Tue, 10 Jan 2012 05:00:00 +0100 5624716 http://www.medworm.com/index.php?rid=5624715&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D22251663%26dopt%3DAbstract We describe the first sandwich enzyme-linked immunosorbent assay (ELISA) capable of recognizing both rat and human activated coagulation Factor XII (FXIIa). Increased plasma concentrations of FXIIa have been associated with adverse outcomes in several cardiovascular disease states. In humans, the FXIIa antigen in plasma is quantified by a direct sandwich ELISA employing an antibody (mAb 2/215) raised against its β-fragment (β-FXIIa), but this assay is unable to detect rat β-FXIIa antigen. Thus, experimental models are at present limited in their capacity to reveal mechanisms by which FXIIa might contribute to cardiovascular pathology. Consistent with overlap between human and rat FXIIa protein epitope sequences, Western blot analysis and ELISA demonstrate that another previously develop... Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=5624715 Tue, 10 Jan 2012 05:00:00 +0100 5624715 http://www.medworm.com/index.php?rid=5604205&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D22245389%26dopt%3DAbstract Authors: Frischmeyer-Guerrerio PA, Schroeder JT Abstract In vitro basophil responses have longed been used in mechanistic studies to help assess the human allergic diathesis, particularly during therapeutic intervention. Recent evidence points to the use of dendritic cells (DCs) in also being valuable in evaluating therapies aimed at lessening disease through immunomodulation. This review article therefore takes a look at some of the recent advances in old and new assays employing both basophils and DCs, with the added perception that the responses mediated by two cell types are insightful towards understanding immune cell mechanisms underlying allergic disease. PMID: 22245389 [PubMed - as supplied by publisher] (Source: Journal of Immunological Methods) Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=5604205 Sun, 08 Jan 2012 05:00:00 +0100 5604205 http://www.medworm.com/index.php?rid=5604206&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D22233878%26dopt%3DAbstract Authors: Stewart A, Liu Y, Lai JR Abstract Monoclonal antibodies are essential reagents for deciphering gene or protein function and have been a fruitful source of therapeutic and diagnostic agents. However, the use of anticarbohydrate antibodies to target glycans for these purposes has been less successful. Glycans contain less hydrophobic functionality than do proteins or nucleic acids, thus individual glycan-antibody interactions are relatively weak. Information encoded by glycans often involves subtle variations of branched oligosaccharides that cannot be detected with conventional antibodies. Here we describe a new phage display selection strategy for identification of high-affinity and specific glycan antibodies. We designed and characterized a phage clone that functionally d... Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=5604206 Sat, 31 Dec 2011 05:00:00 +0100 5604206 http://www.medworm.com/index.php?rid=5578924&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D22226792%26dopt%3DAbstract Authors: Vale AM, Foote JB, Granato A, Zhuang Y, Pereira RM, Lopes UG, Bellio M, Burrows PD, Schroeder HW, Nobrega A Abstract The quantitative simultaneous description of both variable region gene usage and antigen specificity of immunoglobulin repertoires is a major goal in immunology. Current quantitative assays are labor intensive and depend on extensive gene expression cloning prior to screening for antigen specificity. Here we describe an alternative method based on high efficiency single B cell cultures coupled with RT-PCR that can be used for rapid characterization of immunoglobulin gene segment usage, clonal size and antigen specificity. This simplified approach should facilitate the study of antibody repertoires expressed by defined B cell subpopulations, the analysis of i... Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=5578924 Wed, 28 Dec 2011 05:00:00 +0100 5578924 http://www.medworm.com/index.php?rid=5562215&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D22210093%26dopt%3DAbstract Authors: Cayer MP, Samson M, Bertrand C, Dumont N, Drouin M, Jung D Abstract Investigation of the molecular processes which control the development and function of lymphocytes is essential for our understanding of humoral immunity, as well as lymphocyte associated pathogenesis. Adenovirus-mediated gene transfer provided a powerful tool to investigate these processes. We have previously demonstrated that adenoviral vector Ad5/F35 transduces plasma cell lines at a higher efficiency than primary B cells, owing to differences in intracellular trafficking. Given that phosphatases are effectors of intracellular trafficking, here we have analyzed the effects of a panel of phosphatase inhibitors on Ad5/F35 transduction efficiency in B lymphocytes in the present study. FACS analysis was con... Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=5562215 Wed, 21 Dec 2011 05:00:00 +0100 5562215 http://www.medworm.com/index.php?rid=5562214&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D22210094%26dopt%3DAbstract CONCLUSIONS: All retrospective studies can have hidden biases, and thus investigators should not claim new findings before examining the data in detail with the goal of determining whether the findings could be spurious. There were several suspicious findings in our initial analyses: too many analytes found significant, too many very small p-values, a uniformly positive association of analyte level with survival, and a uniformly positive correlation between analyte levels. We were convinced that these findings must be artifacts, and further analyses showed that the findings could be explained by an apparent decrease of analyte level storage time. PMID: 22210094 [PubMed - as supplied by publisher] (Source: Journal of Immunological Methods) Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=5562214 Wed, 21 Dec 2011 05:00:00 +0100 5562214 http://www.medworm.com/index.php?rid=5550957&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D22197974%26dopt%3DAbstract The objective of the AVRP was to determine the feasibility of reducing the number of priming series and booster doses of the licensed Anthrax Vaccine Adsorbed (AVA) (BioThrax®; Emergent BioSolutions, Lansing, MI) and changing the route of administration from subcutaneous (SC) to intramuscular (IM) (Marano et al., 2008). In this paper we report the validation and long term performance characteristics of the assay during its six year application in the AVRP (2002-2008). The critical features are 1) extensive validation of the assay using two standard reference sera; 2) long term stability and 3) consistency of the data for quantitative analysis of human long term anti-PA IgG responses. The reportable value (RV) of the assay was expressed as anti-PA IgG concentration (μg/ml). Accuracy of th... Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=5550957 Sat, 17 Dec 2011 05:00:00 +0100 5550957 http://www.medworm.com/index.php?rid=5550958&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D22178422%26dopt%3DAbstract Authors: Chile N, Evangelista J, Gilman RH, Arana Y, Palma S, Sterling CR, Garcia HH, Gonzalez A, Verastegui M Abstract To fully understand the preliminary stages of Taenia solium oncosphere attachment in the gut, adequate tools and assays are necessary to observe and quantify this event that leads to infection. A fluorescent-based quantitative adhesion assay, using biotinylated activated-oncospheres and monolayers of Chinese hamster ovary cells (CHO-K1) or human intestinal monolayer cells (INT-407, HCT-8 or HT-29), was developed to study initial events during the infection of target cells and to rapidly quantify the in vitro adhesion of T. solium oncospheres. Fluorescein streptavidin was used to identify biotinylated activated-oncospheres adhered to cells. This adherence was quant... Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=5550958 Fri, 09 Dec 2011 05:00:00 +0100 5550958 http://www.medworm.com/index.php?rid=5550960&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D22154892%26dopt%3DAbstract Authors: Alemani D, Pappalardo F, Pennisi M, Motta S, Brusic V Abstract In the last decades the Lattice Boltzmann method (LB) has been successfully used to simulate a variety of processes. The LB model describes the microscopic processes occurring at the cellular level and the macroscopic processes occurring at the continuum level with a unique function, the probability distribution function. Recently, it has been tried to couple deterministic approaches with probabilistic cellular automata (probabilistic CA) methods with the aim to model temporal evolution of tumor growths and three dimensional spatial evolution, obtaining hybrid methodologies. Despite the good results attained by CA-PDE methods, there is one important issue which has not been completely solved: the intrinsic stoc... Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=5550960 Fri, 02 Dec 2011 05:00:00 +0100 5550960 http://www.medworm.com/index.php?rid=5550961&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D22154743%26dopt%3DAbstract In conclusion, the methods investigated in this proof-of-concept study enables for a semi-high-throughput workflow in the screening and investigation of biological active antibodies. PMID: 22154743 [PubMed - as supplied by publisher] (Source: Journal of Immunological Methods) Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=5550961 Thu, 01 Dec 2011 05:00:00 +0100 5550961 http://www.medworm.com/index.php?rid=5550959&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D22155193%26dopt%3DAbstract Authors: Shey MS, Hughes EJ, de Kock M, Barnard C, Stone L, Kollmann TR, Hanekom WA, Scriba TJ Abstract Innate cells are essential for host defense against invading pathogens, and the induction and direction of adaptive immune responses to infection. We developed and optimized a flow cytometric assay that allows measurement of intracellular cytokine expression by monocytes, dendritic cells (DC) and granulocytes, as well as cellular uptake of green-fluorescent protein (GFP)-expressing mycobacteria, in very small volumes of peripheral blood. We show that innate cell stimulation resulted in increased granularity of monocytes and mDC and decreased granulocyte granularity that precluded flow cytometric discernment of granulocytes from monocytes and myeloid DC by forward and side scatter... Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=5550959 Thu, 01 Dec 2011 05:00:00 +0100 5550959 http://www.medworm.com/index.php?rid=5550962&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D22138606%26dopt%3DAbstract Authors: Petrakis I, Mavroeidi V, Stylianou K, Giannakakis K, Daphnis E Abstract One of the most characterized models of murine lupus nephritis is the [NZB×NZW] F1 female hybrid. Extended glomerular IgG deposits may pose an obstacle in studying molecules of interest via indirect immunofluorescence due to secondary antibodies non-specific binding to deposited IgG molecules. Application of Fab fragments may mitigate non-specific interactions in this mouse model. Specifically we provide evidence that blocking paratopic interactions of secondary antibodies with indigenous glomerular IgG deposits is possible. However the blocking effect seems to be related to the species used for secondary antibody production. Increased secondary antibody host species homology with the mouse could make... Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=5550962 Fri, 25 Nov 2011 05:00:00 +0100 5550962 http://www.medworm.com/index.php?rid=5550965&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D22119405%26dopt%3DAbstract In this study, we describe a new helper phage, XP5, which increased the display level of Fab molecules more than two-fold compared to phage rescued with M13KO7. XP5 uses a combination of ribosome binding site spacing alterations and rare codon clusters to reduce the expression of pIII from the helper phage. This reduction in pIII expression leads to an increase in the incorporation of pIII-Fab fusions during phage rescue. The rescued phage displayed a single copy of the Fab molecule, preventing any avidity effects during the selection process. This also suggests that the percentage of the population of phage displaying a Fab molecule is increased when rescued with XP5. Additionally, the phage titers and infectivity are comparable to libraries rescued with M13KO7. After two rounds of pannin... Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=5550965 Sat, 19 Nov 2011 05:00:00 +0100 5550965 http://www.medworm.com/index.php?rid=5550963&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D22123185%26dopt%3DAbstract Authors: Fujimoto A, Kosaka N, Hasegawa H, Suzuki H, Sugano S, Chiba J Abstract Antibody-based drug research involves the preparation of polyclonal and monoclonal antibodies, especially those that are reactive with native G protein-coupled receptors (GPCRs) on the cell membrane. Here, we report that DNA immunization of mice with a plasmid that encodes endothelin A receptor (ETAR) fused to Escherichia coli (E. coli) GroEL at its C-terminus (ETAR-GroEL) induced very strong and specific antibody responses to native ETAR. Co-injection of plasmids that expressed ETAR and GroEL (ETAR+GroEL) induced significantly lower antibody responses compared with the ETAR-GroEL plasmid. Monoclonal antibodies that are prepared by using GroEL as a molecular adjuvant could be used in immunoassays, such ... Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=5550963 Fri, 18 Nov 2011 05:00:00 +0100 5550963 http://www.medworm.com/index.php?rid=5550967&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D22107967%26dopt%3DAbstract Authors: Sroubek J, Krishnan Y, Chinai J, Buhl S, Scharff MD, McDonald TV Abstract We encountered a high degree of clonal hybridoma loss in the course of generating antibodies specific for the hERG potassium channel. A protein that is crucial for controlling heart rhythm, is abundant in parts of the brain and is abnormally expressed in some tumors. Intracellular domains of the protein were used for immunogens and generated adequate antibody responses in mice. Subsequent hybridomas created using Ag8 myeloma fusion partner yielded clones that secreted specific antibody but none could be successfully maintained in culture. A variety of mechanisms, including polyploidy inherent to hybridoma development or production of cytotoxic antibodies, may be responsible for eventual loss of cell ... Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=5550967 Tue, 15 Nov 2011 05:00:00 +0100 5550967 http://www.medworm.com/index.php?rid=5550966&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D22115721%26dopt%3DAbstract Authors: de Oliveira ER, Lima BM, Dos Santos BA, de Moura WC, Nogueira AC Abstract The interferon (IFN) family of cytokines is recognized as a key component of the innate immune response and the first line of defense against viral infection. The usage of the IFN-alpha as a biopharmaceutical has been mainly applied in the treatment of chronic hepatitis C. In the literature it is possible to find a great variety of methods to determine the potency of these cytokines, and many efforts have been made in order to develop practical bioassays to study the biological activity of IFNs. In this technical note, we present a different approach to determine the potency of a recombinant IFN-alpha preparation based on the activation of the signal transducers and activators of transcription 1 (STA... Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=5550966 Sat, 12 Nov 2011 05:00:00 +0100 5550966 http://www.medworm.com/index.php?rid=5550964&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D22119514%26dopt%3DAbstract This report demonstrates how statistically designed experiments could be employed effectively in different stages of a NAb bioassay life cycle in order to characterize, optimize and stabilize the assay with added benefit of resource efficiency. By combining the approach of design of experiments (DOE) with subject matter expertise and experience, we were able to understand thoroughly how assay parameters affect the performance of the assay individually and interactively, identify the key assay parameters, define assay operating ranges and finally achieve a robust and sensitive cell-based assay for the detection of NAbs to Conatumumab. With the goal of developing a cell-based bioassay that is highly optimized for sensitivity, specificity, precision, and robustness, we performed 2 DOE experim... Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=5550964 Sat, 12 Nov 2011 05:00:00 +0100 5550964 http://www.medworm.com/index.php?rid=5550969&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D22100215%26dopt%3DAbstract Authors: Linares EM, Kubota LT, Michaelis J, Thalhammer S Abstract There is an increasing demand for convenient and accurate point-of-care tools that can detect and diagnose different stages of a disease in remote or impoverished settings. In recent years, lateral flow immunoassays (LFIA) have been indicated as a suitable medical diagnostic tool for these environments because they require little or no sample preparation, provide rapid and reliable results with no electronic components and thus can be manufactured at low costs and operated by unskilled personnel. However, even though they have been successfully applied to acute and chronic disease detection, LFIA based on gold nanoparticles, the standard marker, show serious limitations when high sensitivity is needed, such as early... Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=5550969 Thu, 10 Nov 2011 05:00:00 +0100 5550969 http://www.medworm.com/index.php?rid=5550968&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D22100216%26dopt%3DAbstract Authors: Breau C, Cameron DW, Desjardins M, Lee BC Abstract Chancroid, a sexually transmitted genital ulcer disease caused by the Gram-negative bacterium Haemophilus ducreyi, facilitates the acquisition and transmission of HIV. An effective vaccine against chancroid has not been developed. In this preliminary study, the gene encoding the H. ducreyi outer membrane hemoglobin receptor HgbA was cloned into the plasmid pTETnir15. The recombinant construct was introduced into the attenuated Salmonella typhimurium SL3261 strain and stable expression was induced in vitro under anaerobic conditions. The vaccine strain was delivered into the temperature-dependent rabbit model of chancroid by intragastric immunization as a single dose, or as three doses administered at two-weekly intervals. ... Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=5550968 Thu, 10 Nov 2011 05:00:00 +0100 5550968 http://www.medworm.com/index.php?rid=5423455&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D22094241%26dopt%3DAbstract CONCLUSIONS: We describe a simple method for the isolation of functional T cells from semen. Developing standardized methods for processing samples and measuring immunity in the male genital tract may be important in clinical trials of not only candidate HIV-1 vaccines, but in better understanding cellular immunity to a range of sexually transmitted infections of global significance. PMID: 22094241 [PubMed - as supplied by publisher] (Source: Journal of Immunological Methods) Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=5423455 Thu, 10 Nov 2011 05:00:00 +0100 5423455 http://www.medworm.com/index.php?rid=5423456&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D22079255%26dopt%3DAbstract Authors: Hermann RJ, Van der Steen T, Vomhof-Dekrey EE, Benton KD, Failing JJ, Haring JS, Dorsam GP Abstract Vasoactive intestinal peptide receptor-1 signaling in lymphocytes has been shown to regulate chemotaxis, proliferation, apoptosis and differentiation. During T cell activation, VPAC1 mRNA is downregulated, but the effect on its protein levels is less clear. A small number of studies have reported measurement of human VPAC1 by flow cytometry, but murine VPAC1 reagents are unavailable. Therefore, we set out to generate a reliable and highly specific α-mouse VPAC1 polyclonal antibody for use with flow cytometry. After successfully generating a rabbit α-VPAC1 polyclonal antibody (α-mVPAC1 pAb), we characterized its cross-reactivity and showed that it does not recognize other ... Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=5423456 Fri, 04 Nov 2011 04:00:00 +0100 5423456 http://www.medworm.com/index.php?rid=5423458&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D22075274%26dopt%3DAbstract CONCLUSION: This study validated CD80 as the most robust phenotypic marker for human MΦ(IFN-γ), whereas CD200R was upregulated and CD14 was specifically downregulated on MΦ(IL-4). CD163 and CD16 were found to be specific markers for MΦ(IL-10). The GM-CSF/M-CSF differentiation model showed only a partial phenotypic similarity with the IFN-γ/IL-4/IL-10 induced polarization. PMID: 22075274 [PubMed - as supplied by publisher] (Source: Journal of Immunological Methods) Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=5423458 Sat, 29 Oct 2011 04:00:00 +0100 5423458 http://www.medworm.com/index.php?rid=5423457&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D22075275%26dopt%3DAbstract CONCLUSION: This study provides a systematic methodology for MV isolation, identification and quantification, essential for development of MV as diagnostic and prognostic biomarkers of disease. PMID: 22075275 [PubMed - as supplied by publisher] (Source: Journal of Immunological Methods) Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=5423457 Sat, 29 Oct 2011 04:00:00 +0100 5423457 http://www.medworm.com/index.php?rid=5423459&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D22062586%26dopt%3DAbstract In this study we assessed the feasibility of cryopreservation of viable SLN cell samples for flowcytometric analysis, by comparing quantitative analyses of SLN cell samples after freeze-thawing with direct analysis of fresh SLN cell samples. SLN were collected from nine breast cancer patients. From each SLN cell sample, half was used for immediate analysis and half was analyzed after cryopreservation and thawing. Conventional dendritic cell (cDC) and T cell subsets were quantified and phenotypically characterized by flow cytometry. The observed frequencies of both CD1a(+) and CD1a(-)CD11c(+)CD14(-) cDC subsets showed significant correlation between the fresh and frozen-thawed samples. Similar high correlations were found for CD83 and CD86 expression markers on the more frequent (>0.2%) ... Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=5423459 Wed, 26 Oct 2011 04:00:00 +0100 5423459 http://www.medworm.com/index.php?rid=5381980&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D22036924%26dopt%3DAbstract CONCLUSIONS: These data show that HPA-1a antibodies recognise epitopes on αIIbβ3 that are sensitive to EDTA treatment and that cation chelation grossly reduces the sensitivity of the MAIPA assay by diminishing not only HPA-1a alloantibody binding but also 'capture' monoclonal antibody binding. These findings may, in part, explain the current variability in antibody measurement and will guide the development of more sensitive tests for anti-integrin antibodies in NAIT and other conditions. PMID: 22036924 [PubMed - as supplied by publisher] (Source: Journal of Immunological Methods) Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=5381980 Thu, 20 Oct 2011 04:00:00 +0100 5381980 http://www.medworm.com/index.php?rid=5381979&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D22037448%26dopt%3DAbstract CONCLUSIONS: We found an increase in IFNγ production after the activation of HIV-1 specific CD8+ T lymphocytes by histamine (this elevation was blocked by cimetidine), furthermore, we demonstrated a negative correlation between the production of IFNγ and levels of total IgE. PMID: 22037448 [PubMed - as supplied by publisher] (Source: Journal of Immunological Methods) Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=5381979 Thu, 20 Oct 2011 04:00:00 +0100 5381979 http://www.medworm.com/index.php?rid=5381982&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D22033342%26dopt%3DAbstract Authors: Hardy GJ, Lam Y, Stewart SM, Anasti K, Alam SM, Zauscher S Abstract Our work is motivated by the observation that rare, broadly neutralizing antibodies (NAbs), 4E10 and 2F5, associate with HIV-1 lipids as part of a required first step in neutralization before binding to membrane-proximal antigens. Subsequently, induction of these types of NAbs may be limited by immunologic tolerance due to autoreactivity with host cell membranes. Despite the significance of this lipid reactivity there is little experimental evidence detailing NAb-membrane interactions. Simple and efficient screening assays are needed to select antibodies that have similar lipid reactivity as known NAbs. To this end we have developed a surface plasmon resonance (SPR) spectroscopy based assay that monitors a... Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=5381982 Tue, 18 Oct 2011 04:00:00 +0100 5381982 http://www.medworm.com/index.php?rid=5381981&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D22033343%26dopt%3DAbstract Authors: Oliveira T, Resch W, Jankovic M, Casellas R, Nussenzweig MC, Klein IA Abstract Chromosomal translocations require formation and joining of DNA double strand breaks (DSBs). These events disrupt the integrity of the genome and are involved in producing leukemias, lymphomas and sarcomas. Translocations are frequent, clonal and recurrent in mature B cell lymphomas, which bear a particularly high DNA damage burden by virtue of activation-induced cytidine deaminase (AID) expression. Despite the ubiquity of genomic rearrangements, the forces that underlie their genesis are not well understood. Here, we provide a detailed description of a new method for studying these events, translocation capture sequencing (TC-Seq). TC-Seq provides the means to document chromosomal rearrangement... Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=5381981 Tue, 18 Oct 2011 04:00:00 +0100 5381981 http://www.medworm.com/index.php?rid=5381986&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D22019510%26dopt%3DAbstract Authors: Lin S, Houston-Cummings NR, Prinz B, Moore R, Bobrowicz B, Davidson RC, Wildt S, Stadheim TA, Zha D Abstract A fragment of antigen binding (Fab) surface display system was developed using a glycoengineered Pichia pastoris host strain genetically modified to secrete glycoproteins with mammalian mannose-type Man(5)GlcNAc(2) N-linked glycans. The surface display method described here takes advantage of a pair of coiled-coil peptides as the linker while using the Saccharomyces cerevisiae Sed1p GPI-anchored cell surface protein as an anchoring domain. Several Fabs were successfully displayed on the cell surface using this system and the expression level of the displayed Fabs was correlated to that of secreted Fabs from the same glycoengineered host in the absence of the cell wa... Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=5381986 Wed, 12 Oct 2011 04:00:00 +0100 5381986 http://www.medworm.com/index.php?rid=5381985&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D22019643%26dopt%3DAbstract Authors: Ochkur SI, Kim JD, Protheroe CA, Colbert D, Moqbel R, Lacy P, Lee JJ, Lee NA Abstract Mouse models of eosinophilic disorders are often part of preclinical studies investigating the underlying biological mechanisms of disease pathology. The presence of extracellular eosinophil granule proteins in affected tissues is a well established and specific marker of eosinophil activation in both patients and mouse models of human disease. Unfortunately, assessments of granule proteins in the mouse have been limited by the availability of specific antibodies and a reliance on assays of released enzymatic activities that are often neither sensitive nor eosinophil specific. The ability to immunologically detect and quantify the presence of a mouse eosinophil granule protein in biologic... Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=5381985 Wed, 12 Oct 2011 04:00:00 +0100 5381985 http://www.medworm.com/index.php?rid=5381984&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D22019644%26dopt%3DAbstract Authors: Keoshkerian E, Helbig K, Beard M, Zaunders J, Seddiki N, Kelleher A, Hampartzoumian T, Zekry A, Lloyd AR Abstract Hepatitis C virus (HCV)-specific CD4(+) effector T cell responses are likely to play a key role in the immunopathogenesis of HCV infection by promoting viral clearance and maintaining control of viraemia. As the precursor frequency of HCV-specific CD4(+) T cells in peripheral blood is low, favoured assay systems such as intracellular cytokine (ICC) or tetramer staining have limited utility for ex vivo analyses. Accordingly, the traditional lymphocyte proliferation assay (LPA) remains the gold standard, despite detecting responses in only a minority of infected subjects. Recently, we reported development and validation of a novel whole blood CD4(+) effector T ce... Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=5381984 Wed, 12 Oct 2011 04:00:00 +0100 5381984 http://www.medworm.com/index.php?rid=5381983&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D22020291%26dopt%3DAbstract Authors: Gelkop S, Weisman B, Pulak RN, Zharhary D, Isakov N Abstract Signal transduction from the T cell antigen receptor (TCR)/CD3 complex involves six different immunoreceptor tyrosine-based activation motifs (ITAM) located within the cytoplasmic tails of the CD3 chains. Each ITAM possesses two conserved tyrosine residues that can undergo phosphorylation upon TCR/CD3 crosslinking and become a docking site for SH2-containing effector molecules. Specificity of the SH2 domains is determined by their ability to bind a phosphorylated tyrosine in the context of a longer peptide motif within the target protein. As a result, phosphorylation of different tyrosines within the CD3 cytoplasmic tails creates docking sites for distinct SH2-containing signaling proteins that differentially imp... Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=5381983 Wed, 12 Oct 2011 04:00:00 +0100 5381983 http://www.medworm.com/index.php?rid=5381987&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D22004852%26dopt%3DAbstract Authors: Moseley NB, Laur O, Ibegbu CC, Loria GD, Ikwuenzunma G, Jayakar HR, Whitt MA, Altman JD Abstract Detection of antigen-specific T cells at the single-cell level by ELISpot or flow cytometry techniques employing intracellular cytokine staining (ICS) is now an indispensable tool in many areas of immunology. When precisely mapped, optimal MHC-binding peptide epitopes are unknown, these assays use antigen in a variety of forms, including recombinant proteins, overlapping peptide sets representing one or more target protein sequences, microbial lysates, lysates of microbially-infected cells, or gene delivery vectors such as DNA expression plasmids or recombinant vaccinia or adenoviruses expressing a target protein of interest. Here we introduce replication-restricted, recombinan... Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=5381987 Sat, 08 Oct 2011 04:00:00 +0100 5381987 http://www.medworm.com/index.php?rid=5381988&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D22001556%26dopt%3DAbstract Authors: Liao K, Sikkema D, Wang C, Chen K, Dewall S, Lee TN Abstract A cell-based bioassay capable of detecting neutralizing antibodies (NAb) specific to a therapeutic anti-IL-13 monoclonal antibody was developed, validated and used to analyze normal human and asthma serum samples. At the time of this study, a neutralizing assay was unavailable for anti-IL-13 antibody therapeutics with sufficient rigor for validation. Thus, we describe here a method and considerations for validation. The assay used IL-13 responsive HEK293 cells transfected with a secreted embryonic alkaline phosphatase (SEAP) reporter gene. Cells were plated at 5.4×10(4) per assay well due to 90% confluence on the subsequent day. Optimal IL-13 and anti-IL-13 concentrations were determined to be 600pg/mL and 900ng... Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=5381988 Thu, 06 Oct 2011 04:00:00 +0100 5381988 http://www.medworm.com/index.php?rid=5381990&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D21996428%26dopt%3DAbstract Authors: Mbitikon-Kobo FM, Bonneville M, Sekaly RP, Trautmann L Abstract The major function of CD8 T cells is to kill specifically target cells. Moreover in certain incurable diseases, antigen-specific human CD8 T cells are impaired, and assessment of their cytolytic activity could bring insights into their physiopathological role and ways to restore immune dysfunctions for immunotherapeutic purposes. Despite this, T cell cytolytic function has been seldom analyzed thoroughly in humans, due to the lack of approaches well suited for ex vivo assessment of T cell cytotoxicity. Current techniques require prior in vitro expansion of antigen-specific CD8 T cell populations and the use of immortalized cells as targets to measure the cell-mediated killing. Furthermore, bulk cytotoxic activ... Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=5381990 Wed, 05 Oct 2011 04:00:00 +0100 5381990 http://www.medworm.com/index.php?rid=5381989&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D22000977%26dopt%3DAbstract Authors: Papp K, Végh P, Hóbor R, Erdei A, Prechl J Abstract Binding of immunoglobulins and complement fragments to targets of adaptive immune responses can be monitored using collections of arrayed antigens and is used to generate profiles of antibody binding and function. The collection of reliable data on these reactions on a large scale requires the establishment of criteria from sample collection through reaction conditions to normalization strategies. We characterized the detection of IgG, complement C3 and C4 under conditions that better resemble in vivo events than most serological assays and are also relevant for in vitro diagnostic purposes. Immune complex formation was modeled using nitrocellulose-based protein arrays and the effects of factors like anticoagulant use, ... Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=5381989 Wed, 05 Oct 2011 04:00:00 +0100 5381989 http://www.medworm.com/index.php?rid=5381992&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D21989137%26dopt%3DAbstract Authors: Willems CH, Kloosterboer N, Kunzmann S, Kramer BW, Zimmermann LJ, van Iwaarden JF Abstract We were able to demonstrate the presence of transforming growth factor β1 and transforming growth factor β2 (TGF-β1,2) in human as well as porcine pulmonary surfactants and SP-A purified from these surfactants. Human SP-A contained 480±74pg TGF-β1 and 61±16pg TGF-β2 per mg SP-A and human pulmonary surfactant contained 140±28pg TGF-β1 and 67±13 TGF-β2 per mg protein. Porcine SP-A contained 306±46pg TGF-β1 and 43±12pg TGF-β2 per mg SP-A and porcine pulmonary surfactant contained 75±18pg TGF-β1 and 22±13 TGF-β2 per mg protein. Size-exclusion chromatography indicated binding of TGF-β1,2 to SP-A. Deglycosylation of SP-A released TGF-β1,2 from SP-A indicating a role fo... Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=5381992 Sun, 02 Oct 2011 04:00:00 +0100 5381992 http://www.medworm.com/index.php?rid=5381991&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D21996427%26dopt%3DAbstract Authors: Barletta KE, Cagnina RE, Wallace KL, Ramos SI, Mehrad B, Linden J Abstract We developed a flow cytometry-based assay to simultaneously quantify multiple leukocyte populations in the marginated vascular, interstitial, and alveolar compartments of the mouse lung. An intravenous injection of a fluorescently labeled anti-CD45 antibody was used to label circulating and marginated vascular leukocytes. Following vascular flushing to remove non-adherent cells and collection of broncho-alveolar lavage (BAL) fluid, lungs were digested and a second fluorescent anti-CD45 antibody was added ex vivo to identify cells not located in the vascular space. In the naïve mouse lung, we found about 11million CD45+ leukocytes, of which 87% (9.5million) were in the vascular marginated compartmen... Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=5381991 Sun, 02 Oct 2011 04:00:00 +0100 5381991 http://www.medworm.com/index.php?rid=5381995&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D21986105%26dopt%3DAbstract Authors: Rispens T, de Vrieze H, de Groot E, Wouters D, Stapel S, Wolbink GJ, Aarden LA Abstract Rheumatoid factors are antibodies directed against IgG that may confound immunogenicity testing for therapeutic monoclonal antibodies. We developed antigen-binding assays to monitor anti-drug-antibody (ADA) responses against infliximab and adalimumab using F(ab')2 fragments of the drug. This avoids possible detection of rheumatoid factor activity. During development of these assays, a number of sera from patients before treatment as well as several healthy control sera were tested positive. None of these sera contained antibodies specific for the therapeutic mAb. Instead, they were found to contain anti-hinge antibodies. We demonstrate that this aspecific antibody binding can be inhibit... Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=5381995 Sat, 01 Oct 2011 04:00:00 +0100 5381995 http://www.medworm.com/index.php?rid=5381994&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D21986106%26dopt%3DAbstract Authors: Memon SA, Sportès C, Flomerfelt FA, Gress RE, Hakim FT Abstract The analysis of T cell receptor diversity provides a clinically relevant and sensitive marker of repertoire loss, gain, or skewing. Spectratyping is a broadly utilized technique to measure global TCR diversity by the analysis of the lengths of CDR3 fragments in each Vβ family. However the common use of large numbers of T cells to obtain a global view of TCR Vβ CDR3 diversity has restricted spectratyping analyses when limited T-cell numbers are available in clinical setting, such as following transplant regimens. We here demonstrate that one hundred thousand T cells are sufficient to obtain a robust, highly reproducible measure of the global TCR Vβ repertoire diversity among twenty Vβ families in human per... Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=5381994 Sat, 01 Oct 2011 04:00:00 +0100 5381994 http://www.medworm.com/index.php?rid=5381998&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D21767541%26dopt%3DAbstract In this study, we established a new method of Tchreg cell induction, which consists of three steps and provides more efficient and reproducible results. By using a purified T cell population, the efficiency of Tchreg generation was profoundly increased, and the use of purified CD2+ cells rather than CD3+ cells was shown to be superior. One surprising finding was that at the initial step, stromal cell stimulation induced DP T cells more efficiently than dendritic cells or anti-T cell receptor stimulation, indicating a distinct antigen presenting cell (APC) ability of stromal cells as distinguished from conventional APCs. Even in subsequent steps, the presence of stromal cells was required to maintain the DP phenotype. In the second step, addition of stromal cell-conditioned (but not uncondi... Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=5381998 Fri, 30 Sep 2011 04:00:00 +0100 5381998 http://www.medworm.com/index.php?rid=5381997&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D21781971%26dopt%3DAbstract CONCLUSIONS: We showed in various fixed and unfixed cells from different sources that KO is a suitable fluorochrome with a significantly higher quantum yield than PO and is even brighter than other violet laser dyes (e.g. Pacific Blue). CD45-KO/SS enables us to distinguish and characterize various normal and malignant leukocyte subpopulations. By using a 10-color MoAb panel to screen on aberrancies, we showed that CD45-KO provides reliable immunophenotyping within small amounts of cells and thereby improves the quality of 10-color stainings. PMID: 21781971 [PubMed - indexed for MEDLINE] (Source: Journal of Immunological Methods) Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=5381997 Fri, 30 Sep 2011 04:00:00 +0100 5381997 http://www.medworm.com/index.php?rid=5381996&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D21798263%26dopt%3DAbstract Authors: Yathi KK, Joseph JM, Bhasker S, Kumar R, Chinnamma M Abstract Chikungunya is an acute febrile illness caused by an alpha virus technically called as CHIK virus. A smaller size of CHIK virus E1 coat protein -11 kDa was expressed in prokaryotic expression system. The recombinant protein was purified and confirmed by western blot analysis. The positions of the antigenic domain in the protein were identified and the immunoreactivity of recombinant protein with anti-CHIK IgM antibodies was ascertained. The antigen showed an 88% sensitivity and 100% specificity by Indirect ELISA. No cross reactivity of the antigen was observed with anti-Dengue virus serum samples. The results strongly support that the recombinant CHIK coat protein could be used as a diagnostic antigen for the de... Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=5381996 Fri, 30 Sep 2011 04:00:00 +0100 5381996 http://www.medworm.com/index.php?rid=5381993&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D21986107%26dopt%3DAbstract Authors: Zhang GL, Ansari HR, Bradley P, Cawley GC, Hertz T, Hu X, Jojic N, Kim Y, Kohlbacher O, Lund O, Lundegaard C, Magaret CA, Nielsen M, Papadopoulos H, Raghava GP, Tal VS, Xue LC, Yanover C, Zhu S, Rock MT, Crowe JE, Panayiotou C, Polycarpou MM, Duch W, Brusic V PMID: 21986107 [PubMed - as supplied by publisher] (Source: Journal of Immunological Methods) Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=5381993 Thu, 29 Sep 2011 04:00:00 +0100 5381993 http://www.medworm.com/index.php?rid=5298908&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D21968254%26dopt%3DAbstract Authors: Todd CA, Greene KM, Yu X, Ozaki DA, Gao H, Huang Y, Wang M, Li G, Brown R, Wood B, D'Souza MP, Gilbert P, Montefiori DC, Sarzotti-Kelsoe M Abstract Recent advances in assay technology have led to major improvements in how HIV-1 neutralizing antibodies are measured. A luciferase reporter gene assay performed in TZM-bl (JC53bl-13) cells has been optimized and validated. Because this assay has been adopted by multiple laboratories worldwide, an external proficiency testing program was developed to assure data equivalency across laboratories performing this neutralizing antibody assay for HIV/AIDS vaccine clinical trials. The program was optimized by conducting three independent rounds of testing, with an increased level of stringency from the first to third round. Results fro... Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=5298908 Thu, 22 Sep 2011 04:00:00 +0100 5298908 http://www.medworm.com/index.php?rid=5282385&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D21963949%26dopt%3DAbstract Authors: Weiss GE, Ndungu FM, McKittrick N, Li S, Kimani D, Crompton PD, Marsh K, Pierce SK Abstract Memory B cells (MBCs) are a key component of long term humoral immunity to many human infectious diseases. Despite their importance, we know little about the generation or maintenance of antigen-(Ag)-specific MBCs in humans in response to infection. A frequently employed method for quantifying Ag-specific MBCs in human peripheral blood (Crotty et al., 2004) relies on the ability of MBCs but not naïve B cells to differentiate into antibody secreting cells (ASCs) in response to polyclonal activators and Toll-like receptor agonists in vitro and the measurement of Ag-specific ASCs by ELISPOT assays. Here we report on studies to optimize the efficiency of this ELISPOT-based assay and to... Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=5282385 Thu, 22 Sep 2011 04:00:00 +0100 5282385 http://www.medworm.com/index.php?rid=5282384&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D21963950%26dopt%3DAbstract Authors: Malnati MS, Heltai S, Cosma A, Reitmeir P, Allgayer S, Glashoff RH, Liebrich W, Vardas E, Imami N, Westrop S, Nozza S, Tambussi G, Buttò S, Fanales-Belasio E, Ensoli B, Ensoli F, Tripiciano A, Fortis C, Lusso P, Poli G, Erfle V, Holmes H Abstract Delineation of the immune correlates of protection in natural infection or after vaccination is a mandatory step for vaccine development. Although the most recent techniques allow a sensitive and specific detection of the cellular immune response, a consensus on the best strategy to assess their magnitude and breadth is yet to be reached. Within the AIDS Vaccine Integrated Project (AVIP http://www.avip-eu.org) we developed an antigen scanning strategy combining the empirical-based approach of overlapping peptides with a vast arra... Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=5282384 Thu, 22 Sep 2011 04:00:00 +0100 5282384 http://www.medworm.com/index.php?rid=5282389&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D21945394%26dopt%3DAbstract Authors: Schlatzer DM, Sugalski J, Dazard JE, Chance MR, Anthony DD Abstract Dendritic cells (DC) direct the magnitude, polarity and effector function of the adaptive immune response. DC express toll-like receptors (TLR), antigen capturing and processing machinery, and costimulatory molecules, which facilitate innate sensing and T cell activation. Once activated, DC can efficiently migrate to lymphoid tissue and prime T cell responses. Therefore, DC play an integral role as mediators of the immune response to multiple pathogens. Elucidating the molecular mechanisms involved in DC activation is therefore central in gaining an understanding of host response to infection. Unfortunately, technical constraints have limited system-wide 'omic' analysis of human DC subsets collected ex viv... Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=5282389 Fri, 16 Sep 2011 04:00:00 +0100 5282389 http://www.medworm.com/index.php?rid=5282390&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D21945198%26dopt%3DAbstract CONCLUSION: This study provides the proof-of-concept that histamine and its release can be studied by multicolor flow cytometry on a single cell level (HistaFlow). Coupling the data to simultaneous phenotyping of activated basophils confirms that histamine release principally results from anaphylactic degranulation and in a lesser extent from piecemeal degranulation. PMID: 21945198 [PubMed - as supplied by publisher] (Source: Journal of Immunological Methods) Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=5282390 Wed, 14 Sep 2011 04:00:00 +0100 5282390 http://www.medworm.com/index.php?rid=5282387&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D21945396%26dopt%3DAbstract Authors: Esen M, Forster J, Ajua A, Spänkuch I, Paparoupa M, Mordmüller B, Kremsner PG Abstract Immunoglobulin E (IgE) antibodies are major contributors to the pathology of atopic and allergic diseases as well as to immune response to helminth infections. Development of an adequate immunoglobulin G (IgG) immune response against infectious agents and vaccine antigens is considered in most cases as crucial for protection from disease. In vivo and in vitro production of IgE and IgG depends on cytokines and other soluble factors. Recently it has been shown that IgG antibody secreting cells (ASCs) can be generated by in vitro maturation of blood cells with Interleukin- (IL-)15 and CpG DNA or other stimulation cocktails, while IgE-ASCs develop upon cultivation with anti-CD40 and IL-4. ... Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=5282387 Wed, 14 Sep 2011 04:00:00 +0100 5282387 http://www.medworm.com/index.php?rid=5282386&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D21939661%26dopt%3DAbstract Authors: Magistrelli G, Malinge P, Anceriz N, Desmurs M, Venet S, Calloud S, Daubeuf B, Kosco-Vilbois M, Fischer N Abstract The MHC class-I related receptor or neonatal Fc receptor (FcRn) protects IgG and albumin from degradation by rescuing them in endothelial cells in a pH dependent fashion and consequently increases their respective half-lives. Monoclonal antibody-based therapies are of increasing interest and characterizing the interaction with FcRn is important for the development of an antibody candidate. In order to facilitate the production of soluble FcRn suitable for interaction studies, we generated semi-stable pools co-expressing FcRn α-chain, β2-microglobulin, biotin ligase and EGFP using a dual promoter, multi-cistronic vector. Human and mouse FcRn were purified in ... Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=5282386 Mon, 12 Sep 2011 04:00:00 +0100 5282386 http://www.medworm.com/index.php?rid=5282388&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D21945395%26dopt%3DAbstract Authors: Robins H, Desmarais C, Matthis J, Livingston R, Andriesen J, Reijonen H, Carlson C, Nepom G, Yee C, Cerosaletti K Abstract Advances in high-throughput sequencing have enabled technologies that probe the adaptive immune system with unprecedented depth. We have developed a multiplex PCR method to sequence tens of millions of T cell receptors (TCRs) from a single sample in a few days. A method is presented to test the precision, accuracy, and sensitivity of this assay. T cell clones, each with one fixed productive TCR rearrangement, are doped into complex blood cell samples. TCRs from a total of eleven samples are sequenced, with the doped T cell clones ranging from 10% of the total sample to 0.001% (one cell in 100,000). The assay is able to detect even the rarest clones. Th... Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=5282388 Sat, 10 Sep 2011 04:00:00 +0100 5282388 http://www.medworm.com/index.php?rid=5282392&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D21925181%26dopt%3DAbstract Authors: Geering B, Schmidt-Mende J, Federzoni E, Stoeckle C, Simon HU Abstract Neutrophils are terminally differentiated cells with a short life-span due to constitutive apoptosis. Because of these characteristics, genetic manipulation of neutrophils has been difficult, although it is highly desired given the importance of neutrophils in the immune system. Here we demonstrate that transduction of primary human mature neutrophils with enhanced green fluorescent protein (eGFP)-encoding lentiviral particles results in GFP-containing cells as previously reported. Yet, our data further show that GFP expression in neutrophils upon transduction is largely due to protein transfer, a process called lentiviral pseudotransduction, and not due to bona fide transduction. Thus, inhibition of vi... Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=5282392 Thu, 08 Sep 2011 04:00:00 +0100 5282392 http://www.medworm.com/index.php?rid=5282391&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D21945004%26dopt%3DAbstract This study used phospho-flow cytometry as a tool to profile the kinetics and extent of TCR signaling (ZAP-70 and PKC-θ phosphorylation and expression) in Tregs and non-Tregs. We found that in vitro stimulation with anti-CD3ε induces early and transient activation of ZAP-70 and PKC-θ in both Tregs and non-Tregs. However, the response in Tregs was more rapid and higher in magnitude than responses seen in non-Tregs. In contrast, bacterial superantigen or antigen-specific TCR stimulation did not significantly activate these signaling pathways in Tregs or non-Tregs. Additional experiments tested the kinetics of in vivo TCR signaling in Tregs and non-Tregs in mice challenged with bacterial superantigen. The results of these experiments showed that superantigen rapidly activated ZAP-70 and PKC... Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=5282391 Tue, 06 Sep 2011 04:00:00 +0100 5282391 http://www.medworm.com/index.php?rid=5233631&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D21920367%26dopt%3DAbstract In conclusion, 2-AG in cell culture media may significantly influence cellular experiments. CD14+ mononuclear cells which strongly express surface CB receptors may be particularly sensitive towards residual 2-AG from FBS. Therefore, the EC content in culture media should be controlled in biological experiments involving monocytes/macrophages. PMID: 21920367 [PubMed - as supplied by publisher] (Source: Journal of Immunological Methods) Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=5233631 Sat, 03 Sep 2011 04:00:00 +0100 5233631 http://www.medworm.com/index.php?rid=5219712&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D21910993%26dopt%3DAbstract Authors: Lallemand C, Kavrochorianou N, Steenholdt C, Bendtzen K, Ainsworth MA, Meritet JF, Blanchard B, Lebon P, Taylor P, Charles P, Alzabin S, Tovey MG Abstract A cell-based assay has been developed for the quantification of the activity of TNFα antagonists based on human erythroleukemic K562 cells transfected with a NFκB regulated firefly luciferase reporter-gene construct. Both drug activity and anti-drug neutralizing antibodies can be quantified with a high degree of precision within 2h, and without interference from cytokines and other factors known to activate NFκB. The assay cells also contain the Renilla luciferase reporter gene under the control of a constitutive promoter that allows TNFα-induced firefly luciferase activity to be normalized relative to Renilla lucife... Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=5219712 Sat, 03 Sep 2011 04:00:00 +0100 5219712 http://www.medworm.com/index.php?rid=5219714&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D21906599%26dopt%3DAbstract Authors: Hoffman D, Berger M Abstract Most therapeutic proteins induce an unwanted immune response. Antibodies elicited by these therapeutic proteins may significantly alter drug safety and efficacy, highlighting the need for the strategic assessment of immunogenicity at various stages of clinical development. Immunogenicity testing is generally conducted by a multi-tiered approach whereby patient samples are initially screened for the presence of anti-drug antibodies in a screening assay. The screening assay cut point is statistically determined by evaluation of drug-naïve samples and is typically chosen to correspond to a false positive rate of 5%. While various statistical approaches for determination of this screening cut point have been commonly adopted and described in the i... Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=5219714 Thu, 01 Sep 2011 04:00:00 +0100 5219714 http://www.medworm.com/index.php?rid=5219713&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D21907207%26dopt%3DAbstract Authors: Ukaji T, Iwasa MA, Kai O Abstract Hybridomas, generated by fusing myeloma cells with B lymphocytes, secrete monoclonal antibodies (mAbs) specific for an antigen. To establish hybridomas that secrete Mongolian gerbil (Meriones unguiculatus) mAbs, we immunized gerbils with keyhole limpet hemocyanin (KLH) and fused splenocytes from them with a non-secreting mouse myeloma cell line (P3-X63-Ag8.653). We obtained hybridomas that secreted immunoglobulin (Ig) against KLH. These hybridomas had more chromosomes than either parent and retained several gerbil chromosomes. Therefore, these cells were heterohybridomas with both gerbil and mouse chromosomes. They expressed gerbil Ig heavy-chain constant (IGHC) region mRNA, but not mouse gamma (γ) 1 IGHC. SDS-PAGE and Western blot analys... Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=5219713 Wed, 31 Aug 2011 04:00:00 +0100 5219713 http://www.medworm.com/index.php?rid=5219715&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D21903095%26dopt%3DAbstract Authors: Lexmond W, der Mee JV, Ruiter F, Platzer B, Stary G, Yen EH, Dehlink E, Nurko S, Fiebiger E Abstract The aim of this study was to develop a standardized enzyme-linked immunosorbent assay (ELISA) for detection of human soluble Fc-epsilon-RI (sFcεRI), a serum isoform of the high affinity IgE receptor. A recombinant version of sFcεRI was produced in baculovirus and used as standard. ELISA plates were coated with anti-mouse IgG followed by incubation with the monoclonal capture antibody CRA1. This FcεRI-alpha-specific antibody binds to the stalk region of the protein and does not inhibit IgE-binding. After incubation with standards or serum samples, plates were incubated with chimeric IgE followed by detection with horseradish peroxidase conjugated anti-human IgE. Enzymatic... Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=5219715 Sun, 28 Aug 2011 04:00:00 +0100 5219715 http://www.medworm.com/index.php?rid=5219716&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D21893063%26dopt%3DAbstract Authors: López I, Pardo MA Abstract Anisakis simplex has been recognized as an important cause of disease in man and as a foodborne allergen source. Actually, this food-borne was recently identified as an emerging food safety risk including allergenic symptoms. This parasite contains a large variety of allergenic proteins enforcing the necessity to detect new allergens. Commonly, these efforts have been focused on the developing of cDNA libraries, where virtually all expressed mRNAs are present, by using immunoreactive patient serum or polyclonal antibodies. Phage display system is an alternative strategy which permits the physical binding of the genotype with the phenotype, since the products are expressed by the phage on its surface, thereby allowing more efficient selection. In... Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=5219716 Sat, 27 Aug 2011 04:00:00 +0100 5219716 http://www.medworm.com/index.php?rid=5219719&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D21889512%26dopt%3DAbstract This study focused on the latex agglutination immunoassay because it is a simple, homogenous immunoassay, which is also cost effective. We developed a highly sensitive latex reagent and examined the method of antibody conjugation on the latex particle surface. We introduced spacer amino acids in the latex surface to investigate the relationship between the amino acid spacer and the binding of an anti-C-reactive protein (anti-CRP) antibody as well as to investigate the resulting reactivity of the latex reagent to antigen. Because the distance between the latex particle and the antibody is equal in each case, differences in immunoreactivity are attributed to the structure of the amino acid side chain (R). Thus, reactivity of the latex reagent depends on the inorganicity and organicity of R. ... Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=5219719 Fri, 26 Aug 2011 04:00:00 +0100 5219719 http://www.medworm.com/index.php?rid=5219717&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D21893062%26dopt%3DAbstract Authors: Soman G, Yang X, Jiang H, Giardina S, Mitra G Abstract Two assay methods for quantification of the disialoganglioside (GD2)-specific binding activities of anti-GD2 monoclonal antibodies and antibody immunofusion proteins, such as ch14.18 and hu14.18-IL2, were developed. The methods differed in the use of either microtiter plates coated with purified GD2 or plates seeded with GD2-expressing cell lines to bind the anti-GD2 molecules. The bound antibodies were subsequently detected using the reactivity of the antibodies to an HRP-labeled anti-IgG Fc or antibodies recognizing the conjugate IL-2 part of the Hu 14.18IL-2 fusion protein. The bound HRP was detected using reagents such as orthophenylene diamine, 2, 2'-azinobis [3-ethylbenzothiazoline-6-sulfonic acid] or tetramethyl... Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=5219717 Fri, 26 Aug 2011 04:00:00 +0100 5219717 http://www.medworm.com/index.php?rid=5219720&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D21889511%26dopt%3DAbstract Authors: Brubaker AL, Schneider DF, Palmer JL, Faunce DE, Kovacs EJ Abstract Isolation of leukocytes from full-thickness excisional wounds has proven to be a difficult process that results in poor cell yield and holds significant limitations for functional assays. Given the increased interest in the isolation, characterization and functional measurements of wound-derived cell populations, herein we describe a method for preparing wound cell suspensions with an improved yield that enables both phenotypic and functional assessments. PMID: 21889511 [PubMed - as supplied by publisher] (Source: Journal of Immunological Methods) Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=5219720 Thu, 25 Aug 2011 04:00:00 +0100 5219720 http://www.medworm.com/index.php?rid=5219721&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D21889510%26dopt%3DAbstract We present a significant revision of this model which improves the physiological understanding of several parameters. Namely, the parameter used previously as a heuristic explanation for the dilution of CFSE dye by cell division is replaced with a more physical component, cellular autofluorescence. The rate at which label decays is also quantified using a Gompertz decay process. We then demonstrate a revised method of fitting the model to the commonly used histogram representation of the data. It is shown that these improvements result in a model with a strong physiological basis which is fully capable of replicating the behavior observed in the data. PMID: 21889510 [PubMed - as supplied by publisher] (Source: Journal of Immunological Methods) Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=5219721 Wed, 24 Aug 2011 04:00:00 +0100 5219721 http://www.medworm.com/index.php?rid=5219718&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D21889513%26dopt%3DAbstract Authors: Poglitsch M, Katholnig K, Säemann MD, Weichhart T Abstract Due to their low protein content and limited nuclear detergent stability, primary human immune cells such as monocytes or T lymphocytes represent a great challenge for standard nuclear isolation protocols. Nuclei clumping during the multiple centrifugation steps or contamination of isolated nuclei with cytoplasmic proteins due to membrane lysis is a frequently observed problem. Here we describe a versatile and novel method for the isolation of clean and intact nuclei from primary human monocytes, which can be applied for virtually any cell type. Living cells were applied on an iso-osmolar discontinuous iodixanol-based density gradient including a detergent-containing lysis layer. Mild cell lysis as well as efficie... Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=5219718 Wed, 24 Aug 2011 04:00:00 +0100 5219718 http://www.medworm.com/index.php?rid=5219724&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D21878336%26dopt%3DAbstract Authors: Ruwona TB, Johnson VJ, Hettick JM, Schmechel D, Beezhold D, Wang W, Simoyi RH, Siegel PD Abstract Diisocyanates (dNCOs) are highly reactive low molecular weight chemicals used in the manufacture of polyurethane products and are the most commonly reported cause of occupational asthma. Mechanistic disease studies and development of biomonitoring and research tools, such as monoclonal antibodies (mAbs) have been hampered by dNCOs' ability to self-polymerize and to cross-link biomolecules. Toluene diisocyanate (TDI)-specific monoclonal antibodies (mAbs), with potential use in immunoassays for exposure and biomarker assessments, were produced and reactivities characterized against mono- and diisocyanate and dithioisocyanate protein conjugates. In general, TDI reactive mAbs disp... Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=5219724 Mon, 22 Aug 2011 04:00:00 +0100 5219724 http://www.medworm.com/index.php?rid=5219723&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D21878337%26dopt%3DAbstract In this study, we achieved much higher fusion efficiency by stimulating B cells with CpG oligodeoxynucleotide (CpG ODN) over shorter periods. Splenocytes were isolated from immunized mice and cultured in the presence of a CpG ODN for 1 or 2days. This CpG ODN stimulation evokes about one order of magnitude higher fusion efficiency than other stimulators. CpG ODN stimulation not only increases the fusion efficiency but also the number of antibody-producing cells. This leads to a substantial increase in the number of positive clones obtained. This highly efficient fusion method was used to produce a functional antibody against Gaussia luciferase. This method was found to produce greater numbers of hybridomas and to enable direct screening for antibodies with functional characteristics such as... Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=5219723 Mon, 22 Aug 2011 04:00:00 +0100 5219723 http://www.medworm.com/index.php?rid=5219722&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D21878338%26dopt%3DAbstract Authors: Meijerink M, Ulluwishewa D, Anderson RC, Wells JM Abstract Literature on the effects of cryopreservation and thawing of monocytes or monocyte-derived immature dendritic cells (iDCs) on the subsequent functional capacities of the DCs is limited to a few specific maturation stimuli and is focused on applications in clinical immunotherapy. Given the cardinal role of DCs in regulating tolerance and immunity at mucosal surfaces there is a growing interest in understanding the effect of stromal, microbial and probiotic signals on DC function. Therefore our aim was to investigate the effects of cryopreservation on the functional properties of DCs stimulated with bacteria or the bacterial components using a standardized method. Surface markers CD83 and CD86 were expressed at simil... Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=5219722 Mon, 22 Aug 2011 04:00:00 +0100 5219722 http://www.medworm.com/index.php?rid=5179134&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D21872603%26dopt%3DAbstract Authors: Valentino MD, Abdul-Alim CS, Maben ZJ, Skrombolas D, Hensley LL, Kawula TH, Dziejman M, Lord EM, Frelinger JA, Frelinger JG Abstract Epitopes are a hallmark of the antigen specific immune response. The identification and characterization of epitopes is essential for modern immunologic studies, from investigating cellular responses against tumors to understanding host/pathogen interactions especially in the case of bacteria with intracellular residence. Here, we have utilized a novel approach to identify T cell epitopes exploiting the exquisite ability of particulate antigens, in the form of beads, to deliver exogenous antigen to both MHC class I and class II pathways for presentation to T cell hybridomas. In the current study, we coupled this functional assay with two dist... Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=5179134 Wed, 17 Aug 2011 23:00:00 +0100 5179134 http://www.medworm.com/index.php?rid=5179135&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D21871896%26dopt%3DAbstract Authors: Degn SE, Thiel S, Nielsen O, Hansen AG, Steffensen R, Jensenius JC Abstract The lectin pathway of complement is a central part of innate immunity, but as a powerful inducer of inflammation it needs to be tightly controlled. The MASP2 gene encodes two proteins, MASP-2 and MAp19. MASP-2 is the serine protease responsible for lectin pathway activation. The smaller alternative splice product, MAp19, lacks a catalytic domain but retains two of three domains involved in association with the pattern-recognition molecules (PRMs): mannan-binding lectin (MBL), H-ficolin, L-ficolin and M-ficolin. MAp19 reportedly acts as a competitive inhibitor of MASP-2-mediated complement activation. In light of a ten times lower affinity of MAp19, versus MASP-2, for association with the PRMs, much... Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=5179135 Tue, 16 Aug 2011 23:00:00 +0100 5179135 http://www.medworm.com/index.php?rid=5157728&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D21856306%26dopt%3DAbstract Authors: Chan CE, Chan AH, Lim AP, Hanson BJ Abstract Rapid development of diagnostic immunoassays against novel emerging or genetically modified pathogens in an emergency situation is dependent on the timely isolation of specific antibodies. Non-immune antibody phage display libraries are an efficient in vitro method for selecting monoclonal antibodies and hence ideal in these circumstances. Such libraries can be constructed from a variety of sources e.g. B cell cDNA or synthetically generated, and use a variety of antibody formats, typically scFv or Fab. However, antibody source and format can impact on the quality of antibodies generated and hence the effectiveness of this methodology for the timely production of antibodies. We have carried out a comparative screening of two ant... Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=5157728 Thu, 11 Aug 2011 23:00:00 +0100 5157728 http://www.medworm.com/index.php?rid=5142037&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D21846471%26dopt%3DAbstract In conclusion, highly sensitive and equivalent methods were developed and validated to directly compare antibody response to velaglucerase alfa and imiglucerase treatments in patients with Gaucher disease, and may contribute to future internationally standardized assays for antibody detection in patients with Gaucher disease. PMID: 21846471 [PubMed - as supplied by publisher] (Source: Journal of Immunological Methods) Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=5142037 Fri, 05 Aug 2011 23:00:00 +0100 5142037 http://www.medworm.com/index.php?rid=5142039&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D21839739%26dopt%3DAbstract Authors: Bremm M, Huenecke S, Lehrnbecher T, Ponstingl E, Mueller R, Heinze A, Bug G, Quaiser A, Kapinsky M, Brehm C, Bader P, Schneider G, Klingebiel T, Koehl U Abstract Regulatory T cells (Tregs) are of crucial importance to suppress graft versus host disease (GvHD) post allogeneic stem cell transplantation (SCT), but are also known to impair antitumor immunity. However, Treg longitudinal studies are rare and in this respect advanced flowcytometric approaches for Treg characterization are necessary. To investigate the relation of both the percentage and the absolute numbers of Tregs on GvHD or relapse we measured CD4(+)CD25(+/hi)CD127(lo/-) Tregs in 239 peripheral blood (PB) samples of 16 patients during the first two years post-SCT. A 10-color flowcytometric panel was establishe... Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=5142039 Tue, 02 Aug 2011 23:00:00 +0100 5142039 http://www.medworm.com/index.php?rid=5142038&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D21839740%26dopt%3DAbstract In this study, we validated the use of a branched-chain DNA (bDNA) assay known as the QuantiGene Plex 2.0 Reagent System for measuring innate cytokine and chemokine mRNA levels following TLR stimulation of guinea pig cells. Gene expression for T-helper-1 (Th1) polarizing cytokines (TNF-α, IL-1β, IL-12) and chemokines (CXCL1, CCL2) was upregulated following ex vivo stimulation of guinea pig splenocytes and whole blood with TLR-4 or TLR-7/8 agonists. These data confirm the utility of the QuantiGene system both as an alternative to RT-PCR for measuring transcript levels and as a high-throughput screening tool for dissecting the immunological response to TLR stimulation in guinea pigs. Overall, the QuantiGene platform is reliable, reproducible, and sensitive. These agonists have the potentia... Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=5142038 Tue, 02 Aug 2011 23:00:00 +0100 5142038 http://www.medworm.com/index.php?rid=5142040&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D21839083%26dopt%3DAbstract Authors: Dons'koi BV, Chernyshov VP, Osypchuk DV Abstract In present study human peripheral blood NK cell activation after co-incubation with K569 cell line was investigated by CD69 expression. NK lytic activity was studied by two different assays: TDA (2,2':6',2″-terpyridine-6,6″-dicarboxylate) release assay (TRA) and flow cytometry assay (FCA) that display two approach to cytotoxicity measurement. We also investigated NK cell degranulation activity by estimation of CD107a (LAMPa) expression. Comparison of specific lysis value measured by both cytotoxicity assays showed high correlation coefficient between two methods (r=0.94447). Specific lysis value correlated significantly with CD69+ NK frequency and NK degranulation activity. We show that lymphocyte incubation with K562 re... Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=5142040 Mon, 01 Aug 2011 23:00:00 +0100 5142040 http://www.medworm.com/index.php?rid=5142042&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D21835180%26dopt%3DAbstract Authors: Bernth-Jensen JM, Møller BK, Jensenius JC, Thiel S Abstract As much as one percent of antibodies in human plasma are reported to be specific for the non-human disaccharide structure αGal. Various microbes express αGal. However, the implications of anti-αGal antibodies for the anti-microbial defenses are poorly established. With the perspective of studying the biological importance of the antibodies, we have established a sensitive Time-Resolved ImmunoFluorometric Assay (TRIFMA) for quantification of such antibodies. Two versions were developed, one for IgM antibodies and one for IgG antibodies. Samples were collected from plasma donations of healthy adults (n=120) of known gender (60+60), AB0-type (0: 15+15, A: 15+15, B: 15+15, and AB: 15+15) and age (19-64yrs). We sub... Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=5142042 Fri, 29 Jul 2011 23:00:00 +0100 5142042 http://www.medworm.com/index.php?rid=5142043&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D21824477%26dopt%3DAbstract Authors: Hart MH, de Vrieze H, Wouters D, Wolbink GJ, Killestein J, de Groot ER, Aarden LA, Rispens T Abstract The presence of anti-drug antibodies (ADA) in adalimumab-treated patients is associated with reduced serum adalimumab levels and a lower clinical response. Currently, there is no standard for measurement of anti-drug antibodies and many factors influence the results. Consequently, the incidence of ADA as reported in different studies varies considerably. Here we investigated the differential effect of drug interference in two common types of assays used to measure anti-adalimumab: an antigen binding test (ABT) and a more often-used bridging elisa. We measured ADA to adalimumab in a cohort of 216 rheumatoid arthritis patients treated with adalimumab for 28weeks. Only 15 sam... Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=5142043 Thu, 28 Jul 2011 23:00:00 +0100 5142043 http://www.medworm.com/index.php?rid=5142041&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D21835181%26dopt%3DAbstract In conclusion, our results indicate that the Auto 40 system, using thermoresistant reagents, is suitable for CD4 T cell enumeration and will be a helpful tool to improve HIV laboratory monitoring in resource-limited settings. PMID: 21835181 [PubMed - as supplied by publisher] (Source: Journal of Immunological Methods) Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=5142041 Thu, 28 Jul 2011 23:00:00 +0100 5142041 http://www.medworm.com/index.php?rid=5142044&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D21824476%26dopt%3DAbstract Authors: Ten Berge O, van Velsen SG, Giovannone B, Bruijnzeel-Koomen CA, Knol EF, Guikers K, van Weelden H Abstract UV-mediated DNA damage and repair are important mechanisms in research on UV-induced carcinogenesis. UV-induced DNA-damage and repair can be determined by immunohistochemical staining of photoproduct positive nuclei of keratinocytes in the epidermis. We developed a new method of analysing and quantifying thymine dimer (TT-CPD) positive cells in the epidermis. Normal skin of healthy controls was exposed to UVB ex vivo and in vivo. Skin samples were immunohistochemically stained for TT-CPDs. Digital images of the epidermis were quantified for TT-CPDs both visually and digitally. There was a UVB-dose dependent induction of TT-CPDs present in the ex vivo UVB-irradiated sk... Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=5142044 Wed, 27 Jul 2011 23:00:00 +0100 5142044 http://www.medworm.com/index.php?rid=5142046&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D21821036%26dopt%3DAbstract Authors: Song L, Hanlon DW, Chang L, Provuncher GK, Kan CW, Campbell TG, Fournier DR, Ferrell EP, Rivnak AJ, Pink BA, Minnehan KA, Patel PP, Wilson DH, Till MA, Faubion WA, Duffy DC Abstract The quantitative measurement of inflammatory cytokines in blood has been limited by insufficient sensitivity of conventional immunoassays. This limitation has prevented the widespread clinical monitoring of cytokine concentrations in chronic inflammatory diseases. We applied a sensitive, single molecule detection technology to measure TNF-α and IL-6 in the plasma of patients with Crohn's disease (CD), before and after treatment with anti-TNF-α therapy. Plasma from 17 patients with CD was collected prior to initiation of anti-TNF-α therapy, and the Crohn's disease activity index (CDAI) was de... Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=5142046 Tue, 26 Jul 2011 23:00:00 +0100 5142046 http://www.medworm.com/index.php?rid=5142045&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D21821037%26dopt%3DAbstract CONCLUSIONS: The results suggest that this minimally invasive, rapid and sensitive assay will be useful in large patient screening studies for risk assessment of coronary events. PMID: 21821037 [PubMed - as supplied by publisher] (Source: Journal of Immunological Methods) Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=5142045 Tue, 26 Jul 2011 23:00:00 +0100 5142045 http://www.medworm.com/index.php?rid=5106628&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D21798263%26dopt%3DAbstract Authors: Yathi KK, Joseph JM, Bhasker S, Kumar R, Chinnamma M Chikungunya is an acute febrile illness caused by an alpha virus technically called as CHIK virus. A smaller size of CHIK virus E1 coat protein -11kDa was expressed in prokaryotic expression system. The recombinant protein was purified and confirmed by western blot analysis. The positions of the antigenic domain in the protein were identified and the immunoreactivity of recombinant protein with anti-CHIK IgM antibodies was ascertained. The antigen showed an 88% sensitivity and 100% specificity by Indirect ELISA. No cross reactivity of the antigen was observed with anti-Dengue virus serum samples. The results strongly support that the recombinant CHIK coat protein could be used as a diagnostic antigen for the detection of Chi... Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=5106628 Wed, 20 Jul 2011 23:00:00 +0100 5106628 http://www.medworm.com/index.php?rid=5106639&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D21782818%26dopt%3DAbstract Authors: Camper N, Byrne T, Burden RE, Lowry J, Gray B, Johnston JA, Migaud ME, Olwill SA, Buick RJ, Scott CJ Monoclonal antibodies and derivative formats such as Fab fragments are used in a broad range of therapeutic, diagnostic and research applications. New systems and methodologies that can improve the production of these proteins are consequently of much interest. Here we present a novel approach for the rapid production of processed Fab' fragments in a CHO cell line that has been engineered to express the mouse cationic amino acid transporter receptor 1 (m-CAT1). This facilitated the introduction of the target antibody gene through retroviral transfection, rapidly producing stable expression. Using this system, we designed a single retroviral vector construct for the expression o... Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=5106639 Tue, 19 Jul 2011 23:00:00 +0100 5106639 http://www.medworm.com/index.php?rid=5106632&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D21787778%26dopt%3DAbstract CONCLUSIONS: TRACP is an important marker in osteoimmunology. Specific antibodies with unique specificity for TRACP isoforms and defined applications will be valuable for clinical evaluation of bone metabolic, inflammatory and autoimmune diseases and will aid in basic research of TRACP biochemistry and biology. PMID: 21787778 [PubMed - as supplied by publisher] (Source: Journal of Immunological Methods) Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=5106632 Tue, 19 Jul 2011 23:00:00 +0100 5106632 http://www.medworm.com/index.php?rid=5106631&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D21787779%26dopt%3DAbstract CONCLUSIONS: Absolute CD4+ T-cell counts and percent generated using capillary blood are in close agreement with those from venous blood. Point-Of-Care assays and standard flow cytometers can be deployed in a tiered laboratory network where both venous and capillary blood collection can be used for CD4+ T-cell enumeration. PMID: 21787779 [PubMed - as supplied by publisher] (Source: Journal of Immunological Methods) Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=5106631 Tue, 19 Jul 2011 23:00:00 +0100 5106631 http://www.medworm.com/index.php?rid=5106630&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D21787780%26dopt%3DAbstract Authors: Niu L, Guo C, Hao Z, Yuan J, Li Z The open reading frame of the α-subunit (amino acids 1-211) of human muscle nicotinic acetylcholine receptor (hAChR(α211)) was inserted into eukaryotic expression vector of pPIC9K to form a recombinant plasmid. After transformation and expression, the target protein rhAChR(α211) (recombinant hAChR(α211)) was secretively expressed in recombinant strain P. pastoris GS115/pPIC9K-hAChR(α211) with a yield of 25mg/L. rhAChR(α211) was purified by Q Sepharose column and gel filtration chromatography. Furthermore, the purified protein was coupled with CNBr-actived Sepharose 4B to form a special immunoadsorbent. By this immunoadsorbent, the removal rate for AChRAb in two myasthenia gravis (MG) patient sera reached 84% and 94%, respectively. The DN... Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=5106630 Tue, 19 Jul 2011 23:00:00 +0100 5106630 http://www.medworm.com/index.php?rid=5106629&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D21787781%26dopt%3DAbstract Authors: Kim HS, Lo SC, Wear DJ, Stojadinovic A, Weina PJ, Izadjoo MJ To improve anti-Burkholderia monoclonal antibody (MAb) binding affinity, six single chain variable fragments (scFvs) constructed previously were used as scaffolds to construct large highly-diversified phage-displayed mouse scFv random and domain libraries. First, we employed random mutagenesis to introduce random point mutations into entire variable regions, generating six random libraries. Additionally, the oligonucleotide-directed mutagenesis was targeted on complementarity-determining region 3 (CDR3) from each variable region of heavy (VH) and light chains (VL) derived from six scFvs, and generated eighteen domain libraries including six VH CDR3, six VL CDR3, and six combined VH/VL CDR3 mutated domains, respective... Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=5106629 Tue, 19 Jul 2011 23:00:00 +0100 5106629 http://www.medworm.com/index.php?rid=5106640&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D21781972%26dopt%3DAbstract Authors: Ruitenberg JJ, Boyce C, Hingorani R, Putnam A, A Ghanekar S Human regulatory T cells (Treg) are able to actively suppress autoreactive immune responses. Phenotypically, they are broadly characterized as CD4(+), CD25(+), CD127(lo/-) and FoxP3(+). CD45RA can be used to further differentiate the population into naïve (CD45RA(+)) and induced (CD45RA(-)) Treg. The functional potential of Treg is routinely determined by assessing their ability to suppress T cell function in 3-5day proliferation assays. Since Treg are being explored for therapeutic use, a short-term functional assay could serve as a valuable tool for evaluating the potency of Treg. Therefore, an assay designed to measure Treg suppression of activation marker expression by responder T cells in 7 to 20h has been exami... Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=5106640 Mon, 18 Jul 2011 23:00:00 +0100 5106640 http://www.medworm.com/index.php?rid=5106633&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D21784078%26dopt%3DAbstract In this study, we optimized a flow cytometry-based assay to study intracellular expression levels of three important host proteins involved in HIV-1 replication: apolipoprotein B mRNA-editing catalytic polypeptide-like 3G (APOBEC3G), tripartite motif 5alpha (TRIM5α), and lens epithelium-derived growth factor (LEDGF/p75). An indirect intracellular staining (ICS) method was optimized using antibodies designed for other applications like enzyme-linked immunosorbent assay (ELISA), confocal imaging, and western blotting. The median fluorescence intensity (MFI) value - a measure for the protein expression level - increased upon higher antibody concentration and longer incubation time, and was reduced following preincubation with recombinant proteins. Staining of stably transfected or knock-down... Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=5106633 Mon, 18 Jul 2011 23:00:00 +0100 5106633 http://www.medworm.com/index.php?rid=5106641&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D21781970%26dopt%3DAbstract Authors: Dabitao D, Margolick JB, Lopez J, Bream JH Serum cytokine profiling is a powerful tool to link host immune defense with disease pathogenesis. Although several multiplex assays are commercially available, none has been rigorously validated in the context of chronic infectious disease (such as HIV infection). Here we compared the measurement of proinflammatory cytokines by two multiplex platforms: the Meso Scale Discovery (MSD) electrochemiluminescence assay and the Becton Dickinson Cytometric Bead Array (CBA) flow cytometric assay, using serum samples from HIV-infected and -uninfected donors. We evaluated the ability of these assays to: a) quantify circulating levels of native cytokines (IL-6, IL-8, IL-10, TNF-α, IL-12p70, IL-1β), and b) accurately recover known amounts of re... Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=5106641 Sun, 17 Jul 2011 23:00:00 +0100 5106641 http://www.medworm.com/index.php?rid=5106638&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D21782819%26dopt%3DAbstract Authors: Massilamany C, Gangaplara A, Chapman N, Rose N, Reddy J A/J mice bearing the H-2 allele IA(k) are highly susceptible to autoimmune myocarditis induced with cardiac myosin heavy chain (Myhc)-α 334-352, whereas B10.A mice carrying a similar allele IA(k) are relatively resistant, suggesting that the generation of Myhc-α-reactive T cell repertoires is influenced by genetic background. To enumerate the precursor frequencies of Myhc-α-specific CD4 T cells, we sought to create IA(k) tetramers for Myhc-α 334-352. Tetramers were created using approaches that involve covalent tethering of individual peptide sequences or exogenous loading of peptides into empty IA(k) molecules by peptide-exchange reaction. Using ribonuclease 43-56 tetramers as controls, we demonstrated that by flow c... Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=5106638 Sun, 17 Jul 2011 23:00:00 +0100 5106638 http://www.medworm.com/index.php?rid=5106637&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D21782820%26dopt%3DAbstract Authors: Zhang GL, Lin HH, Keskin DB, Reinherz EL, Brusic V The immune system is characterized by high combinatorial complexity that necessitates the use of specialized computational tools for analysis of immunological data. Machine learning (ML) algorithms are used in combination with classical experimentation for the selection of vaccine targets and in computational simulations that reduce the number of necessary experiments. The development of ML algorithms requires standardized data sets, consistent measurement methods, and uniform scales. To bridge the gap between the immunology community and the ML community, we designed a repository for machine learning in immunology named Dana-Farber Repository for Machine Learning in Immunology (DFRMLI). This repository provides standardized d... Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=5106637 Sun, 17 Jul 2011 23:00:00 +0100 5106637 http://www.medworm.com/index.php?rid=5106636&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D21782821%26dopt%3DAbstract Authors: Chang C, Takayanagi A, Yoshida T, Shimizu N We recently constructed the scFv-displaying phage library with extremely high repertoire and have successfully utilized for screening scFv antibodies against various proteins, polysaccharides and glyco-lipids. Here, we developed a new screening strategy to isolate scFv antibodies against cell surface EGF receptor (EGFR). For this, we applied two slightly different methods of "target-guided proximity labeling," such as Proximity selection (ProxiMol) method and a new sulfo-SBED labeling method with the aide of monoclonal anti-human EGFR antibody B4G7 as a guide molecule. ProxiMol method relies on the Biotin-labeling of scFv-displaying phages that bound to the target in a vicinity of 100Å from the guide molecule, whereas sulfo-SBED me... Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=5106636 Sun, 17 Jul 2011 23:00:00 +0100 5106636 http://www.medworm.com/index.php?rid=5106635&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D21782822%26dopt%3DAbstract In conclusion, mechanical tissue disaggregation by Medimachine™ appears to be optimal to maintain phenotypic and functional TIIC features. PMID: 21782822 [PubMed - as supplied by publisher] (Source: Journal of Immunological Methods) Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=5106635 Sun, 17 Jul 2011 23:00:00 +0100 5106635 http://www.medworm.com/index.php?rid=5106634&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D21782823%26dopt%3DAbstract Authors: Xing D, Markey K, Newland P, Rigsby P, Hockley J, He Q As part of the new EUVAC.NET contract with ECDC (Pertussis Work Area 4), a collaborative study was organised in July-December 2010. Two well-defined reference preparations with high and low IgG antibodies to pertussis toxin (PT), were sent to participants. The purposes of this study were to assess current laboratory performance of serological assays for pertussis; to compare in-house reference preparations that are currently used by participants for the serological assay; and to identify needs for standardisation of the serological assay. Reference Laboratories in Europe currently performing serological assays for the diagnosis of pertussis by measuring antibody to PT, were invited to participate in the study. A total of 1... Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=5106634 Sun, 17 Jul 2011 23:00:00 +0100 5106634 http://www.medworm.com/index.php?rid=5060517&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D21777590%26dopt%3DAbstract Authors: Chen X, Zhuang Y, Wang Q, Moutsoglou D, Ruiz G, Yen SE, Dreskin SC It is important to know the contribution of specific allergens to a complex allergenic extract and to have a dependable method to assess the effector activity of an extract specifically depleted of that allergen. We have previously shown that removal of the major peanut allergen, Ara h 2, from a crude peanut extract (CPE) minimally altered the effector activity of the extract. Here we describe in detail the methodology used to generate specific rabbit anti-peptide antibodies to remove a related peanut allergen, Ara h 6, from CPE and describe an improvement in the RBL SX-38 cell assay used to assess the effector activity of treated extracts. Our results show that although Ara h 2 and Ara h 6 can be selectively r... Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=5060517 Sun, 17 Jul 2011 23:00:00 +0100 5060517 http://www.medworm.com/index.php?rid=5060516&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D21777591%26dopt%3DAbstract Authors: Makvandi-Nejad S, Fjällman T, Arbabi-Ghahroudi M, Mackenzie CR, Hall JC Three V(H)Hs against the model hapten, azoxystrobin (MW 403) were isolated from a hyper-immunized phage-displayed V(H)H library. This library was constructed by isolating the V(H)H-coding genes from the lymphocytes collected from a Llama glama that was immunized with azoxystrobin conjugated to bovine serum albumin (BSA). Six rounds of panning were performed against azoxystrobin conjugated to either ovalbumin (OVA) or rabbit serum albumin (RSA) to enrich clones containing V(H)Hs specific to the hapten. After screening 95 clones, three V(H)Hs (A27, A72, and A85) with different amino acid sequences were identified, expressed in soluble format in Escherichia coli HB2151, and purified using nickel-immobilized ... Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=5060516 Sun, 17 Jul 2011 23:00:00 +0100 5060516 http://www.medworm.com/index.php?rid=5060515&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D21777592%26dopt%3DAbstract Authors: Liu L, Johnson C, Fujimura S, Teque F, Levy JA Electroporation, a non-virus-mediated gene transfection method, has traditionally had poor outcomes with low gene transfection efficiency and poor cellular viability, particularly in primary human lymphocytes. Herein we have optimized the electroporation conditions for primary CD8+ cells resulting in a maximum rate of 81.3%, and a mean transfection efficiency of 59.6%. After removal of dead cells, the viability of transfected primary CD8+ cells was greater than 90%, similar to untransfected controls. Using this procedure, primary human CD8+ cells transfected with an interferon α8 plasmid produced fluids that inhibited HIV-1 replication by >95%. This transfection protocol is useful for transfection of other primary blood cells,... Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=5060515 Sun, 17 Jul 2011 23:00:00 +0100 5060515 http://www.medworm.com/index.php?rid=5060520&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D21767540%26dopt%3DAbstract Authors: Neaga A, Lefor J, Lich KE, Liparoto SF, Xiao YQ Phagocytosis plays a pivotal and essential role in host immune defense, both as a focal constituent of the innate immune response and a bridging element linking innate and adaptive immunity. Phagocytosis has been demonstrated to be critical in development, tissue remodeling, wound healing and resolution of inflammation through clearance of foreign organisms, apoptotic cells and the production of anti-inflammatory mediators. During pre-clinical investigations, therapeutic drug candidates may alter host resistance to infectious agents by modulating the immune system. The assessment of phagocytic function can be a critical parameter of immunotoxicology for this adverse effect. Utilizing pH-sensitive pHrodo™ BioParticles®, a flow ... Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=5060520 Thu, 14 Jul 2011 23:00:00 +0100 5060520 http://www.medworm.com/index.php?rid=5060519&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D21767541%26dopt%3DAbstract In this study, we established a new method of Tchreg cell induction, which consists of three steps and provides more efficient and reproducible results. By using a purified T cell population, the efficiency of Tchreg generation was profoundly increased, and the use of purified CD2(+) cells rather than CD3(+) cells was shown to be superior. One surprising finding was that at the initial step, stromal cell stimulation induced DP T cells more efficiently than dendritic cells or anti-T cell receptor stimulation, indicating a distinct antigen presenting cell (APC) ability of stromal cells as distinguished from conventional APCs. Even in subsequent steps, the presence of stromal cells was required to maintain the DP phenotype. In the second step, addition of stromal cell-conditioned (but not unc... Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=5060519 Thu, 14 Jul 2011 23:00:00 +0100 5060519 http://www.medworm.com/index.php?rid=5060521&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D21763696%26dopt%3DAbstract Authors: Shi C, Belisle D, Miller JD Aspergillus versicolor, a fungus commonly found on damp building materials, produces the allergen, Asp v 13. Here we report a sensitive Asp v 13 capture ELISA for A. versicolor spores and spore- and mycelial fragments in house dust samples. The method is based on a double polyclonal capture ELSIA. The detection limits for Asp v 13 antigen and A. versicolor spores without dust were 2.44pg and 12ng (ca. 110 spores). Detection limits for Asp v 13 and A. versicolor spores in sieved house dust samples were 1.0ng and 7.8μg per gram dry weight house dust, respectively. This detection limit is lower than for other house dust allergen immunoassays including for Stachybotrys chartarum, Aspergillus fumigatus, but much lower than that from Alternaria alternata... Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=5060521 Wed, 13 Jul 2011 23:00:00 +0100 5060521 http://www.medworm.com/index.php?rid=5060518&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D21771595%26dopt%3DAbstract We report a recently encountered example of the latter resulting in interference in a solid-phase sandwich assay. Due to the set-up employing a monoclonal rat IgG for capture and a monoclonal rat IgM for development the interference had to be human antibodies reacting with rat light-chain. Out of 102 Danish Caucasian blood donors we find a prevalence of anti-rat kappa light chain antibodies of close to 40% (39/102, defined as at least 2-fold elevated measurements), with around 6% (6/102) having very high levels (defined as at least 4-fold elevated measurements), yielding significantly higher measurements in the assay designed to measure the complement component MAp19 in serum samples. The interference could be blocked by the addition of rat immunoglobulin to the sample buffer. An individua... Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=5060518 Mon, 11 Jul 2011 23:00:00 +0100 5060518 http://www.medworm.com/index.php?rid=5060525&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D21749873%26dopt%3DAbstract Authors: Soderstrom CI, Spriggs FP, Sun W, Burrell S Several detection platforms are available for ligand binding assays (LBA), each claiming superiority in sensitivity and dynamic range. However, little information exists in the literature directly comparing the various LBA platforms for quantitation. We have tested four common platforms to evaluate and compare the interchangeability of detection platforms by comparing sensitivity and dynamic range to a colorimetric LBA. The detection platforms compared are: colorimetric, chemiluminescence, time-resolved fluorescence (TRF) and electrochemiluminescence (ECL). Five different LBA protocols were tested with each of the detection endpoints. The assay protocols include the following ligand binding assay formats: direct binding, sandwich ELI... Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=5060525 Sat, 02 Jul 2011 23:00:00 +0100 5060525 http://www.medworm.com/index.php?rid=5060526&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D21741978%26dopt%3DAbstract CONCLUSION: To detect allergen-specific T(H) cells for a common allergen as grass-pollen, expansion is not absolutely necessary, although within 8-day grass-pollen cultures, higher numbers of proliferating cells resulted in increased statistical power to detect phenotypic differences. However, this approach also detects more bystander activated T(H) cells. TCLs resulted in comparable percentages of cytokine expressing T cells as 8-day cultures. Therefore enrichment can be necessary for detection of T(H) cells specific for a single allergen or allergen-derived peptide, but is dispensable for the detection and phenotyping of allergen-specific T(H) cells using crude extracts. PMID: 21741978 [PubMed - as supplied by publisher] (Source: Journal of Immunological Methods) Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=5060526 Fri, 01 Jul 2011 23:00:00 +0100 5060526 http://www.medworm.com/index.php?rid=5060529&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D21741383%26dopt%3DAbstract Authors: Findlay L, Eastwood D, Ball C, Robinson CJ, Bird C, Wadhwa M, Thorpe SJ, Thorpe R, Stebbings R, Poole S Two methods for predicting the risk of pro-inflammatory clinical infusion reactions during monoclonal antibody therapy were evaluated. In the first, the antibody of interest is immobilised by air-drying onto 96-well plates prior to the addition of human peripheral blood mononuclear cells (PBMCs). In the second, the antibody is added in aqueous phase to a co-culture of human PBMCs and human endothelium-derived cells. In both methods the cells are incubated with the antibody to allow the accumulation of pro-inflammatory cytokines, quantified by enzyme-linked immunosorbent assay (ELISA). The antibodies associated with clinical infusion reactions, Herceptin, Campath-1H and TGN14... Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=5060529 Wed, 29 Jun 2011 23:00:00 +0100 5060529 http://www.medworm.com/index.php?rid=5060528&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D21741384%26dopt%3DAbstract This study describes a novel filtration method to isolate PMVs in plasma, which avoids high speed centrifugation, and to quantify them using a Becton Dickinson(BD) FACS Calibur™ flow cytometer, as annexin V-positive vesicles, larger than 0.2μm in diameter. Essentially microvesicles (which comprise a mixture of PMVs and exosomes) from citrate plasma were sonicated to break up clumped exosomes, and filtered using Millipore 0.1μm pore size Hydrophilic Durapore membranes in Swinnex 13mm filter holders. Phosphatidylserine-positive PMVs detected with annexin V-PE were quantified using combined labelling and gating strategies in conjunction with Polysciences Polybead Microspheres (0.2μm) and BDTrucount tubes. The PMV absolute count was calculated on the analysis template using the Trucount t... Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=5060528 Wed, 29 Jun 2011 23:00:00 +0100 5060528 http://www.medworm.com/index.php?rid=5060527&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D21741385%26dopt%3DAbstract In this study, we have isolated, by phage display, an sdAb against CEACAM6 with an affinity of 5nM from a llama immunized with cancer cells. The antibody has good biophysical properties, and it binds to the cells expressing the target antigen. Furthermore, it reduces cancer cells proliferation in vitro and shows an excellent tumor targeting in vivo. This sdAb could be useful in diagnosis as well as therapy of CEACAM6 expressing tumors. Finally, we envisage it would be feasible to isolate good sdAbs against other interesting tumor associated antigens from this library. Therefore, this immunization method could be a general strategy for isolating sdAbs against any surface antigen without immunizing the animal with the antigen of interest each time. PMID: 21741385 [PubMed - as supplied by... Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=5060527 Wed, 29 Jun 2011 23:00:00 +0100 5060527 http://www.medworm.com/index.php?rid=5060523&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D21756911%26dopt%3DAbstract Authors: Kellner C, Bleeker WK, Lammerts van Bueren JJ, Staudinger M, Klausz K, Derer S, Glorius P, Muskulus A, de Goeij BE, van de Winkel JG, Parren PW, Valerius T, Gramatzki M, Peipp M Antibody-drug conjugates (ADC) represent promising agents for targeted cancer therapy. To allow rational selection of human antibodies with favorable characteristics for ADC development a screening tool was designed obviating the need of preparing individual covalently linked conjugates. Therefore, α-kappa-ETA' was designed as a fusion protein consisting of a human kappa light chain binding antibody fragment and a truncated version of Pseudomonas exotoxin A. α-kappa-ETA' specifically bound to human kappa light chains of human or human-mouse chimeric antibodies and Fab fragments. Antibody-redirected �... Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=5060523 Wed, 29 Jun 2011 23:00:00 +0100 5060523 http://www.medworm.com/index.php?rid=4963537&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D21689658%26dopt%3DAbstract This study shows that following target cell interaction there is a substantial loss of NK cells, the extent of which correlates with measures of NK cell cytotoxicity assessed by the target cell release of (51)Cr and by the externalisation of the lysosomal marker LAMP-1 (CD107a) which is assessed on the remaining NK cells. This is the case for the killing of K562 (natural killing) and the CD20 mAb (Rituximab)-mediated killing of RAJI cells and autologous B cells (antibody-dependent cell cytotoxicity). This target-induced NK loss (TINKL) provides a sensitive and specific measure of NK cell responses appropriate to a clinical laboratory setting. PMID: 21689658 [PubMed - as supplied by publisher] (Source: Journal of Immunological Methods) Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=4963537 Thu, 16 Jun 2011 23:00:00 +0100 4963537 http://www.medworm.com/index.php?rid=4963535&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D21693123%26dopt%3DAbstract Authors: Lemmon JC, McFarland RJ, Rybicka JM, Balce DR, McKeown KR, Krohn RM, Matsunaga TO, Yates RM The professional phagocytes, such as macrophages and dendritic cells, are the subject of numerous research efforts in immunology and cell biology. The use of primary phagocytes in these investigations however, are limited by their inherent resistance to transfection with DNA constructs. As a result, the use of phagocyte-like immortalized cell lines is widespread. While these cell lines are transfection permissive, they are generally regarded as poor biological substitutes for primary phagocytes. By exploiting the phagocytic machinery of primary phagocytes, we developed a non-viral method of DNA transfection of macrophages that employs intraphagosomal sonoporation mediated by internalize... Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=4963535 Mon, 13 Jun 2011 23:00:00 +0100 4963535 http://www.medworm.com/index.php?rid=4963536&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D21689659%26dopt%3DAbstract Authors: Fofana IB, Colantonio AD, Reeves RK, Connole MA, Gillis JM, Hall LR, Sato S, Audin CR, Evans DT, Shida H, Johnson RP, Johnson WE SIV infection of macaques is the most widely employed model for preclinical AIDS vaccine and pathogenesis research. In macaques, high-titer virus-specific antibodies are induced by infection, and antibody responses can drive evolution of viral escape variants. However, neutralizing antibodies (Nabs) induced in response to SIVmac239 and SIVmac251 infection or immunization are generally undetectable or of low titer, and the identification and cloning of potent Nabs from SIVmac-infected macaques remains elusive. Based on recent advances in labeling HIV-specific B lymphocytes [1-3], we have generated recombinant, secreted, soluble SIVmac envelope (Env) p... Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=4963536 Sun, 12 Jun 2011 23:00:00 +0100 4963536 http://www.medworm.com/index.php?rid=4963554&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D21664360%26dopt%3DAbstract Authors: Terwee JA, Chin CL, Watrin S, Tello RF, Rieder NJ, Lowell JD, Latham-Timmons D The use of cells as a cryopreserved, readily available reagent has facilitated high-throughput screening of new drug candidates by bioassay. This practice is considerably less labor intensive and allows more flexibility in laboratory testing than traditional continuous cell culture. We have shown that this practice can be adapted to cell proliferation and reporter gene assay formats used in routine sample testing for determination of relative potency of commercial product in a Quality Control Laboratory. The ability to use the same, optimized population of cells provides consistency in an assay over time. Measures of assay performance to indicate maintenance of the validated state of a method and to... Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=4963554 Sat, 11 Jun 2011 23:00:00 +0100 4963554 http://www.medworm.com/index.php?rid=4963538&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D21669205%26dopt%3DAbstract Authors: Kaltsidis H, Cheeseman H, Kopycinski J, Ashraf A, Cox MC, Clark L, Anjarwalla I, Dally L, Bergin P, Spentzou A, Higgs C, Gotch F, Gazzard B, Gomez R, Hayes P, Kelleher P, Gill DK, Gilmour J The next generation of candidate HIV vaccines include replicating vectors selected for tropism to mucosal sites, where an efficacious T cell response will be required to limit T cell replication and HIV associated CD4 T cell loss. To fully assess immunogenicity of such candidates, there is a need to develop robust quality controlled analysis of gut derived HIV specific CD8+ T-cell responses. Despite obvious challenges in obtaining sufficient amounts of tissue, the highly compartmentalised nature of the mucosal immune responses, requires the assessment of CD8 T cells isolated directly from l... Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=4963538 Sat, 11 Jun 2011 23:00:00 +0100 4963538 http://www.medworm.com/index.php?rid=4818381&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D21447345%26dopt%3DAbstract In this study, an immunoquantification assay for human and recombinant IDS was developed with the combination of two antibodies. Rabbit IgG and chicken IgY were used as IDS capture and detection antibodies, respectively. Chicken IgY antibodies were developed against specific amino acid sequences present in IDS but absent in other human sulfatases. hrIDS produced in P. pastoris, commercial hrIDS, and normal human plasma samples were used as antigens and immunoquantification results were compared to enzyme activity. The technique was linear over the range 8 to 500ngmL(-1) using commercial hrIDS. The concentration range detected for IDS in normal human plasma was 14.43 to 287.88ngmL(-1). The hrIDS was detected in P. pastoris cultures even when the enzyme was inactive, which is convenient for ... Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=4818381 Fri, 13 May 2011 18:01:23 +0100 4818381 http://www.medworm.com/index.php?rid=4818380&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D21453706%26dopt%3DAbstract Authors: Bosshart H, Heinzelmann M In the continuous presence of bacterial lipopolysaccharide (LPS), the membrane-bound LPS receptor, CD14, exhibits a biphasic pattern of surface expression in monocytes ex vivo; an initial increase is followed by a later decrease. In the presence of LPS, ex vivo changes in monocytic CD14 cell surface expression have been consistently interpreted as the direct result of LPS exposure. There has been little consideration for the possibility of additional cell culture effects. Here, an experiment is presented to dissect the differences between LPS effects and cell culture effects on monocytic CD14 cell surface expression ex vivo. The results show that in monocytes from diluted whole blood cultures, CD14 surface expression is induced in LPS-treated samples ... Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=4818380 Fri, 13 May 2011 18:01:14 +0100 4818380 http://www.medworm.com/index.php?rid=4818371&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D21554885%26dopt%3DAbstract In conclusion, we have developed a sensitive, specific and direct immunoassay for fPSA-N which can be used to study the clinical relevance of this PSA isoform. PMID: 21554885 [PubMed - as supplied by publisher] (Source: Journal of Immunological Methods) Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=4818371 Wed, 27 Apr 2011 23:00:00 +0100 4818371 http://www.medworm.com/index.php?rid=4818373&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D21530531%26dopt%3DAbstract Authors: Eng HS, Leffell MS Histocompatibility testing has been used in support of solid organ and hematopoietic stem cell transplantation for over fifty years and transplantation has clearly served as a major stimulus for interest in the human major histocompatibility complex, the HLA system. Until the 1990s, typing and definition of antibodies to HLA antigens was performed primarily by serologic techniques using cell-based assays. Two major technological advances have greatly increased knowledge of HLA alleles and HLA-specific antibodies, namely the introduction of DNA based molecular typing and solid phase immunoassays using purified HLA antigens as targets. By virtue of these advances, the number of recognized HLA alleles has increased from a few hundred to greater than 6000 and de... Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=4818373 Sat, 16 Apr 2011 23:00:00 +0100 4818373 http://www.medworm.com/index.php?rid=4818374&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D21513715%26dopt%3DAbstract CONCLUSION: CD4(+)CD39(+) Treg capture on immunobeads led to a discovery of two CD39(+) subsets. Similar to CD39(+) Treg in the peripheral blood, half of these cells are CD25(+)FOXP3(+) active suppressor cells, while the other half are CD25(neg)FOXP3(neg) and do not mediate suppression. PMID: 21513715 [PubMed - as supplied by publisher] (Source: Journal of Immunological Methods) Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=4818374 Tue, 12 Apr 2011 23:00:00 +0100 4818374 http://www.medworm.com/index.php?rid=4818377&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D21497162%26dopt%3DAbstract Authors: Usami Y, Matsuda K, Sugano M, Ishimine N, Kurihara Y, Sumida T, Yamauchi K, Tozuka M In atherosclerotic artery walls, mast cells, an inflammatory cell, are activated and secrete some proteases including chymase. Chymase, a chymotrypsin-like protease, cleaves the C-terminus of apolipoprotein A-I (apoA-I) at Phe225. This cleavage reduces the ability of apoA-I to promote the efflux of cellular cholesterol. The aim of this study is to detect C-terminally truncated apoA-I in normal human serum. For this purpose, we generated a monoclonal antibody that specifically recognizes C-terminally truncated apoA-I by immunizing mice with a peptide that corresponds to human apoA-I amino acid residues 216-225. The monoclonal antibody, termed 16-4mAb, selectively reacted with recombinant C-term... Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=4818377 Fri, 08 Apr 2011 23:00:00 +0100 4818377 http://www.medworm.com/index.php?rid=4818376&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D21501617%26dopt%3DAbstract Authors: Haney D, Quigley MF, Asher TE, Ambrozak DR, Gostick E, Price DA, Douek DC, Betts MR Current technology to isolate viable cytokine-producing antigen-specific primary human T cells is limited to bi-specific antibody capture systems, which suffer from limited sensitivity and high background. Here, we describe a novel procedure for isolating antigen-specific human T cells based on their ability to produce tumor necrosis factor (TNF)-α. Unlike many cytokines, TNF-α is initially produced in a biologically active membrane-bound form that is subsequently cleaved by TNF-α converting enzyme (TACE) to release the soluble form of TNF-α. By preventing this cleavage event, we show that TNF-α can be 'trapped' on the surface of the T cells from which it originates and directly labeled fo... Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=4818376 Thu, 07 Apr 2011 23:00:00 +0100 4818376 http://www.medworm.com/index.php?rid=4818375&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D21501618%26dopt%3DAbstract Authors: Desplancq D, Rinaldi AS, Stoessel A, Sibler AP, Busso D, Oulad-Abdelghani M, Van Regenmortel MH, Weiss E In spite of their many potential applications, recombinant antibody molecules selected by phage display are rarely available commercially, one reason being the absence of robust bacterial expression systems that yield sufficient quantities of reagents for routine applications. We previously described the construction and validation of an intrabody library that allows the selection of single-chain Fv (scFv) fragments solubly expressed in the cytoplasm. Here, we show that it is possible to obtain monomeric scFvs binding specifically to human papillomavirus type 16 E6 and cellular gankyrin oncoproteins in quantities higher than 0.5g/L of shake-flask culture in E. coli cytoplas... Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=4818375 Thu, 07 Apr 2011 23:00:00 +0100 4818375 http://www.medworm.com/index.php?rid=4818378&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D21477591%26dopt%3DAbstract Authors: Matos CS, Coelho-Dos-Reis JG, Rassi A, Luquetti AO, Dias JC, Eloi-Santos SM, Gomes IT, Vitelli-Avelar DM, Wendling AP, Rocha RD, Teixeira-Carvalho A, Peruhype-Magalhães V, Andrade MC, Martins-Filho OA One of the challenges on immunodiagnostic of Chagas disease in endemic areas has been the search for more practical and safe antigenic preparation that provides tests with higher sensitivity and specificity, with low cross-reactivity. A new approach using fixed Trypanosoma cruzi epimastigotes to detect IgG reactivity was investigated previously. In order to continue this investigation, this study aimed at optimizing the flow cytometry-based method to the diagnosis of Chagas disease patients after specific chemotherapy. To achieve our goal, serum samples from 93 subjects - 52 adu... Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=4818378 Tue, 05 Apr 2011 23:00:00 +0100 4818378 http://www.medworm.com/index.php?rid=4818379&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D21466808%26dopt%3DAbstract Authors: Breslin WL, Strohacker K, Carpenter KC, Haviland DL, McFarlin BK Monocytes have been used to assess immune dysfunction and disease. While mouse models are a useful longitudinal analog, few researchers have assessed changes in mouse monocytes. The purpose of this study was to provide recommendations for the sample processing and flow cytometric analysis of mouse blood monocytes. Blood was drawn in a non-lethal manner from CD-1 male mice to be used in three experiments. Experiment 1 compared commonly used mouse monocyte markers. Experiment 2 compared the stability of CD115 expression after immediate (0h) and delayed (2 and 4h) processing following blood collection under various experimental conditions (laser strength, anticoagulant, and storage temp.). Experiment 3 compared the ... Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=4818379 Fri, 01 Apr 2011 23:00:00 +0100 4818379 http://www.medworm.com/index.php?rid=4657915&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D21420412%26dopt%3DAbstract We report a new method for the direct imaging and quantification of ADCC of cancer cells. The proposed method using imaging flow cytometry combines the statistical power of flow cytometry with the analytical advantages of cell imaging, providing a novel and more comprehensive perspective of effector/target cell interactions during ADCC events. With this method we can quantify and show in detail the morphological changes in target and effector cells, their apoptotic index, the physical interaction between effector and target cells, and a directional transfer of cytosolic contents from effector to target cells. As a model system we used the therapeutic anti-CD20 antibody rituximab to target CFSE labeled Ramos human Burkitt's lymphoma cells, to CMTPX-labeled human monocytic U937 effector cell... Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=4657915 Mon, 21 Mar 2011 00:00:00 +0100 4657915 http://www.medworm.com/index.php?rid=4594863&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D21385585%26dopt%3DAbstract Authors: Baum PD, Young JJ, McCune JM T cell receptor (TCR) diversity is critical for adaptive immunity. Existing methods for measuring such diversity are qualitative, expensive, and/or of uncertain accuracy. Here, we describe a method and associated reagents for estimating the absolute number of unique TCR Vβ rearrangements present in a given number of cells or volume of blood. Compared to next generation sequencing, this method is rapid, reproducible, and affordable. Diversity of a sample is calculated based on three independent measurements of one Vβ-Jβ family of TCR rearrangements at a time. The percentage of receptors using the given Vβ gene is determined by flow cytometric analysis of T cells stained with anti-Vβ family antibodies. The percentage of receptors using the Vβ g... Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=4594863 Sat, 05 Mar 2011 00:00:00 +0100 4594863 http://www.medworm.com/index.php?rid=4594864&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D21376053%26dopt%3DAbstract Authors: Zhang H, Wang S, Fang G The recent development and application of multi-analyte simultaneous analysis by enzyme-linked immunosorbent assays (ELISAs) of many kinds of organic compounds are reviewed herein. Multi-analyte ELISAs based on generic antibodies directly, or on specific antibodies by multi-antibody co-coating or multi-tracer co-marking are described in detail. Special attention was placed on the design and synthesis of a generic hapten, the manner of linking the hapten to a carrier protein, type and character of the antibody and format of the ELISAs. It is hoped that this comprehensive review can be considered as a useful reference for further investigation of multi-analyte simultaneous analysis by ELISAs. PMID: 21376053 [PubMed - as supplied by publisher] (Source:... Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=4594864 Tue, 01 Mar 2011 00:00:00 +0100 4594864 http://www.medworm.com/index.php?rid=4594866&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D21362427%26dopt%3DAbstract Authors: Petrunkina AM, Harrison RA Many research projects in cell biology now use flow cytometry for analysis or for isolation of specific cell types. In such studies, cell viability is obviously a crucial issue. However, many studies appear to rely upon light-scattering characteristics to identify and gate out non-viable cells, despite the fact that reliable identification of such cells can only be achieved through staining with impermeable fluorescent nuclear dyes such as propidium iodide or 7-amino actinomycin. In this paper we apply mathematical analysis to the theoretical problem of quantifying cell sub-populations labeled with two or more fluorescent markers, comparing situations in which dead cells have been identified with those in which cell viability has not been assessed. W... Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=4594866 Sat, 26 Feb 2011 00:00:00 +0100 4594866 http://www.medworm.com/index.php?rid=4594865&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D21362428%26dopt%3DAbstract Authors: Memic A, Volgina VV, Gussin HA, Pepperberg DR, Kay BK To generate monoclonal antibodies to the human ρ1 GABA(C) receptor, a ligand-gated chloride ion channel that is activated by the neurotransmitter γ-aminobutyric acid (GABA), we recovered the immunoglobulin variable heavy chain (V(H)) and light chain (V(L)) regions of a guinea pig immunized with a 14-mer peptide segment of the N-terminal extracellular domain of the ρ1 subunit. Oligonucleotide primers were designed and used to amplify the V(H) and V(L) regions of guinea pig RNA by the reverse transcriptase polymerase chain reaction. The amplified and cloned V(H) and V(L) regions were transferred together into a phagemid vector, yielding a library of 5×10(6) members, which displayed chimeric fragments of antigen binding (F... Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=4594865 Sat, 26 Feb 2011 00:00:00 +0100 4594865 http://www.medworm.com/index.php?rid=4534934&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D21354161%26dopt%3DAbstract Authors: McLelland BT, Gravano D, Castillo J, Montoy S, Manilay JO Thymic epithelial cells (TECs) are microenvironmental niche cells which support T lymphocyte development in the thymus. Most studies of TEC biology have focused on TEC at the fetal stage of development, whereas the biology of adult-stage TECs is not as well-understood. Delineating the molecular mechanisms that control adult TEC differentiation has implications for the success of T-lymphocyte based therapies for autoimmune diseases and induction of immunological tolerance to stem cell-derived tissues. Detailed analysis of adult TECs is technically challenging due to their rarity, their diminishing numbers with age, and the limited number of markers to distinguish between unique TEC subpopulations. Here, we have devised a... Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=4534934 Wed, 23 Feb 2011 00:00:00 +0100 4534934 http://www.medworm.com/index.php?rid=4534933&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D21354162%26dopt%3DAbstract Authors: Fernandes VC, Coitinho JB, Veloso JM, Araújo SA, Pedroso EP, Goes AM Paracoccidioidomycosis (PCM) is one of the most important endemic mycoses in Latin America; it's usually diagnosed by observation and/or isolation of the etiologic agent, Paracoccidioides brasiliensis, as well as by a variety of immunological methods, such as complement fixation and immunodiffusion. Although these approaches are useful, historically their sensitivity and specificity have often been compromised by the use of complex mixtures of undefined antigens. The use of combinations of purified, well-characterized antigens appears preferable and may yield good results. In the present study combinations of the previously described 27-kDa recombinant antigen (rPb27) and a recombinant 40-kDa-molecular-mass ... Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=4534933 Wed, 23 Feb 2011 00:00:00 +0100 4534933 http://www.medworm.com/index.php?rid=4534932&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D21354163%26dopt%3DAbstract Authors: Nizet Y, Gillet L, Schroeder H, Lecuivre C, Louahed J, Renauld JC, Gianello P, Vanderplasschen A Antigen expression and purification are laborious, time consuming and frequently difficult steps in the process of antibody production. In the present study, we developed a method avoiding these two steps. This method relies on the injection of histocompatible living cells stably expressing the antigen as a cell surface type II transmembrane fusion protein. A vector, nicknamed pCD1-CD134L, was constructed to express the antigen fused at the carboxyterminal end of the human CD134 ligand (CD134L) type II transmembrane protein on the surface of eucaryotic cells. This vector was shown to induce cell surface expression of epitopes from human c-Myc (soluble protein), uterogloblin-related... Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=4534932 Wed, 23 Feb 2011 00:00:00 +0100 4534932 http://www.medworm.com/index.php?rid=4534937&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D21334341%26dopt%3DAbstract Authors: Lemaire MM, Vanhaudenarde A, Nizet Y, Dumoutier L, Renauld JC Induction of autoantibodies towards immune regulatory proteins, such as cytokines or their receptors, is a powerful strategy for functional studies on the role of these factors in vivo. Here we describe a new procedure to elicit autoantibodies by taking advantage of tumor cells as a vaccine against peptides presented at their surface in fusion with the human CD134L transmembrane protein. P1.HTR, an immunogenic variant of the P815 mastocytoma cell line, was used to generate stably transfected cell clones with expression vectors encoding the human CD134L transmembrane protein fused with either mouse IL-22BP or IL-9. Following repeated injections of living tumor cells expressing the mIL-22BP construct, mice developed a... Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=4534937 Thu, 17 Feb 2011 00:00:00 +0100 4534937 http://www.medworm.com/index.php?rid=4534936&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D21334342%26dopt%3DAbstract Authors: Wadhwa M, Bird C, Hamill M, Heath AB, Matejtschuk P, Thorpe R, Five candidate preparations of human sequence recombinant granulocyte-colony stimulating factor (G-CSF) were formulated and lyophilized at NIBSC prior to evaluation in a collaborative study for their suitability to serve as a replacement International Standard (IS). The preparations were tested by 13 laboratories using in vitro bioassays. The candidate preparation 09/136 was judged suitable to serve as a replacement international standard based on the data obtained for biological activity and stability. On the basis of the results reported here, the preparation coded 09/136 was established by the WHO Expert Committee on Biological Standardization (ECBS) as the 2nd IS for human G-CSF with an assigned value for G-CS... Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=4534936 Thu, 17 Feb 2011 00:00:00 +0100 4534936 http://www.medworm.com/index.php?rid=4534935&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D21334343%26dopt%3DAbstract In conclusion, these new assays will greatly improve the use of the hamster as an important small animal model in infectious disease research. PMID: 21334343 [PubMed - as supplied by publisher] (Source: Journal of Immunological Methods) Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=4534935 Thu, 17 Feb 2011 00:00:00 +0100 4534935 http://www.medworm.com/index.php?rid=4534938&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D21324321%26dopt%3DAbstract Authors: Liebenberg LJ, Gamieldien H, Mkhize NN, Jaumdally SZ, Gumbi PP, Denny L, Passmore JA Cervical cytobrushing, biopsy, or lavages have previously been used to collect mononuclear cells from the female genital tract. Compared with blood, obtaining cells from the female genital tract is more invasive and generally yields few cells for subsequent immune studies. Because of the value of including mucosal sampling in HIV vaccine trials, standardisation of methods for collection, processing, and analysis of immunity from cells derived from the female genital tract is important. The aim of this study was to assess the effect of transport conditions on the viability, recovery and antigenic responsiveness of cervical T cells. This was investigated in cervical cytobrush specimens collected... Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=4534938 Sat, 12 Feb 2011 00:00:00 +0100 4534938 http://www.medworm.com/index.php?rid=4482185&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D21315722%26dopt%3DAbstract Authors: Ren D, Darlucio MR, Chou JH The detection of low level of protein A leached from monoclonal antibody downstream purification process is often interfered by the presence of excess amount of product antibody. In order to prevent this interference, we developed a new multi-product leached protein A assay that used acidification to completely dissociate the IgG-ProteinA complex, followed by neutralization under selected condition to prevent re-formation of the IgG-ProteinA complex. The amount of protein A was then determined by a sandwich immunoassay using Meso Scale Discovery technology. The assay takes approximately 3 hours to complete for one 96-well plate of samples, and this has been successfully applied to samples containing different monoclonal antibody products examined so... Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=4482185 Wed, 09 Feb 2011 00:00:00 +0100 4482185 http://www.medworm.com/index.php?rid=4482184&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D21315723%26dopt%3DAbstract Authors: Reeves RK, Evans TI, Gillis J, Wong FE, Connole M, Carville A, Johnson RP Since the vast majority of infections occur at mucosal surfaces, accurate characterization of mucosal immune cells is critically important for understanding transmission and control of infectious diseases. Standard flow cytometric analysis of cells obtained from mucosal tissues can provide valuable information on the phenotype of mucosal leukocytes and their relative abundance, but does not provide absolute cell counts of mucosal cell populations. We developed a bead-based flow cytometry assay to determine the absolute numbers of multiple mononuclear cell types in colorectal biopsies of rhesus macaques. Using 10-color flow cytometry panels and pan-fluorescent beads, cells were enumerated in biopsy specim... Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=4482184 Wed, 09 Feb 2011 00:00:00 +0100 4482184 http://www.medworm.com/index.php?rid=4482186&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D21296083%26dopt%3DAbstract Authors: Grimberg BT Historically, examinations of the inhibition of malaria parasite growth/invasion, whether using drugs or antibodies, have relied on the use of microscopy or radioactive hypoxanthine uptake. These are considered gold standards for measuring the effectiveness of antimalarial treatments, however, these methods have well known shortcomings. With the advent of flow cytometry coupled with the use of fluorescent DNA stains allowed for increased speed, reproducibility, and qualitative estimates of the effectiveness of antibodies and drugs to limit malaria parasite growth which addresses the challenges of traditional techniques. Because materials and machines available to research facilities are so varied, different methods have been developed to investigate malaria parasit... Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=4482186 Fri, 04 Feb 2011 00:00:00 +0100 4482186 http://www.medworm.com/index.php?rid=4482187&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D21295038%26dopt%3DAbstract Authors: Theorell J, Schlums H, Chiang SC, Huang TY, Tattermusch A, Wood SM, Bryceson YT An early step in immunosurveillance by cytotoxic lymphocytes is leukocyte functional antigen (LFA)-1-dependent adhesion to target cells, which is promoted by inside-out signals from receptors such as the T cell receptor and a variety of natural killer (NK) cell activating receptors. Inside-out signals induce a conformational change in LFA-1, resulting in an extension of the extracellular domain of the receptor. Here, we have evaluated several mAbs that specifically detect the extended conformation of LFA-1 and detail a protocol for flow cytometric quantification of β2-integrin activation in human peripheral blood cytotoxic T cells and NK cells in response to target cell recognition. By comparison ... Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=4482187 Wed, 02 Feb 2011 00:00:00 +0100 4482187 http://www.medworm.com/index.php?rid=4482194&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D21277302%26dopt%3DAbstract In this study, STa peptide of enterotoxigenic Escherichia coli K99(+) was purified and successfully covalently cross-linked to modified bovine serum albumin after thorough evaluation of three different hapten-carrier conjugation protocols. Dimethyformamide (DMF) based STa-conjugation protocol demonstrated higher biological activity (10×10(6) STa Total Mouse Units [MU]) and 100% conjugation efficiency. A range of conjugation ratio of 4-12 STa molecules per one molecule of BSA was achieved and confirmed by matrix-assisted laser desorption ionization-time of flight/mass spectroscopy (MALDI-TOF/MS). This conjugate was used for immunization of ten rabbits for STa antibody production. A high antibody binding titer (10(6)) against STa was obtained with a neutralization capacity of 3×10(4) STa M... Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=4482194 Fri, 28 Jan 2011 00:00:00 +0100 4482194 http://www.medworm.com/index.php?rid=4482193&cid=s_33859_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fentrez%2Fquery.fcgi%3Ftmpl%3DNoSidebarfile%26db%3DPubMed%26cmd%3DRetrieve%26list_uids%3D21277303%26dopt%3DAbstract Authors: Huttunen R, Soini J, Härkönen P, Hänninen P, Härmä H A luminometric method for quantitative cell surface protein expression analysis has been developed in a microtiter plate format. The method is based on immunocytochemistry, the use of long-lived europium(III) and terbium(III) chelates and platinum(II) porphyrin luminescence labels in addition to short-lived syto13 DNA stain, and detection of photoluminescence emission from adhered cells by both time-resolved luminescence and conventional fluorescence. After the immunoreactions, the wells were evaporated to dryness, allowing repeated and postponed luminescence analysis even after months and cellular protein localization studies by microscopy imaging. The multiparametric method assayed the cell surface expression of ß1-i... Journal of Immunological Methods journals http://www.medworm.com/rss/comments.php?id=4482193 Fri, 28 Jan 2011 00:00:00 +0100 4482193