Journal of Immunological MethodsThis is an RSS file. You can use it to subscribe to this data in your favourite RSS reader, such as GoogleReader, or to display this data on your own website or blog. Subscribe to this data using MyMedWorm.Subscribe to this data using GoogleReader.Subscribe to this data using Bloglines.Subscribe to this data using MyYahoo.

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Preparing serum for functional complement assays.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
To conserve full complement activity, a number of precautions need to be taken in preparing serum. This is particularly the case with mouse complement where the classical pathway is notoriously unstable. PMID: 19909755 [PubMed - as supplied by publisher] (Source: Journal of Immunological Methods)
Source: Journal of Immunological Methods - November 10, 2009 Category: Allergy & Immunology Authors: Lachmann PJ Tags: J Immunol Methods Source Type: journals

Improvement of a recombinant antibody-based serological assay for foot-and-mouth disease virus.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
In this study, we have determined the precise binding site of the recombinant detecting antibody to a conserved sequence within the 3B region of the 3ABC protein, replaced the original E-tag of the detecting antibody with two in-house tags and engineered a direct antibody-reporting enzyme (alkaline phosphatase) fusion protein. These modifications have further improved the DIVA test, providing great potential for large scale production and uptake due to its simplicity, reproducibility and low cost. PMID: 19913020 [PubMed - as supplied by publisher] (Source: Journal of Immunological Methods)
Source: Journal of Immunological Methods - November 10, 2009 Category: Allergy & Immunology Authors: Muller JD, Wilkins M, Foord AJ, Dolezal O, Yu M, Heine HG, Wang LF Tags: J Immunol Methods Source Type: journals

Assessment of chemokine receptor function on monocytes in whole blood: In vitro and ex vivo evaluation of a CCR2 antagonist.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
We describe an assay to assess chemokine receptor function on whole blood monocytes by measuring chemokine stimulated change in cell shape as measured by flow cytometry. The relative potential of the chemokine receptors CCR1, CCR2, CCR5, CX(3)CR1, and CXCR4 to activate monocytes in whole blood was evaluated and compared. Analysis of MCP-1 response for monocytes in blood from numerous donors revealed that the assay method had excellent intra-donor reproducibility and sensitivity. Further, the utility of this assay to determine target engagement by chemokine receptor antagonists was demonstrated using a CCR2 antagonist in rh...
Source: Journal of Immunological Methods - November 10, 2009 Category: Allergy & Immunology Authors: Wisniewski T, Bayne E, Flanagan J, Shao Q, Wnek R, Matheravidathu S, Fischer P, Forrest MJ, Peterson L, Song X, Yang L, Demartino JA, Struthers M Tags: J Immunol Methods Source Type: journals

A defined serum-free medium useful for monitoring anti-melanoma responses induced by dendritic cell immunotherapy.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Animal sera provide a non-defined source of nutrients and growth factors for mammalian cell culture. Animal serum supplementation may also introduce experimental artefacts, including immune responses against foreign serum proteins. This artefact is particularly apparent in tumour immunotherapy experiments using dendritic cells (DC) and melanoma cells cultured in fetal calf serum (FCS)-replete media. FCS culture of both DC and melanoma cells significantly enhanced anti-tumour responses in mice immunized with DC that had not been pulsed with tumour antigen. Although serum free media (SFM) may be used for short term cultu...
Source: Journal of Immunological Methods - November 7, 2009 Category: Allergy & Immunology Authors: Bouwer AL, Netter P, Kemp RA, McLellan AD Tags: J Immunol Methods Source Type: journals

Identification and Characterization of HLA-A*0301 epitopes in HIV-1 gag proteins using a Novel Approach.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
In this study we used a new approach to identify and characterize potential epitopes in the gag region containing PS mutations that significantly correlated with HLA-A*0301. The iTopia Epitope Discovery System was used to rapidly screen a panel of peptides overlapping the regions containing PS mutations and the peptides identified were assessed for relative affinity and complex stability. The potential epitopes were then validated by interferon gamma (IFN-gamma) ELISpot assays with patient PBMCs. Using this approach we identified/confirmed the predicted HLA-A*0301 epitopes in two regions of gag containing PS mutations V7I ...
Source: Journal of Immunological Methods - November 7, 2009 Category: Allergy & Immunology Authors: Semeniuk CA, Capina RE, Mendoza MG, Kimani J, Ball TB, Luo M, Plummer FA Tags: J Immunol Methods Source Type: journals

Improved in vitro methods to predict the in vivo toxicity in man of therapeutic monoclonal antibodies including TGN1412.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
TGN1412 is a "superagonistic" CD28 monoclonal antibody (IgG4) that caused serious adverse events at its first time in man clinical trial. In the present study, different in vitro methods for detecting and quantifying unwanted pro-inflammatory activity of therapeutic monoclonal antibodies (mAbs) such as TGN1412 are described. The antibody of interest is immobilised by wet-coating or air-drying onto polypropylene or polystyrene 96-well plates prior to the addition of human peripheral blood mononuclear cells (PBMCs). The cells are incubated for 16 - 24h with the immobilised antibody which allows the accumulation of pro-in...
Source: Journal of Immunological Methods - November 3, 2009 Category: Allergy & Immunology Authors: Findlay L, Eastwood D, Stebbings R, Sharp G, Mistry Y, Ball C, Hood J, Thorpe R, Poole S Tags: J Immunol Methods Source Type: journals

Amplification of immunoassays using phage-displayed single domain antibodies.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Single domain antibodies (sdAb) are recombinantly produced variable domains derived from the heavy-chain only antibodies found in camelids. Previously, we selected sdAb that were specific for both ricin and botulinum A (BoNT A) toxin complex from phage display libraries of sdAb and evaluated the solubly expressed protein. Here, phage displayed sdAb were used as reporter reagents and compared to soluble, unfused sdAb. We found that using phage displayed sdAb as reporter elements in immunoassay formats gave improved detection over using unfused, soluble sdAb reporters. In enzyme-linked immunosorbent assays (ELISAs), the ...
Source: Journal of Immunological Methods - November 3, 2009 Category: Allergy & Immunology Authors: Goldman ER, Anderson GP, Bernstein RD, Swain MD Tags: J Immunol Methods Source Type: journals

A novel method to clean protein G agarose for affinity column matrix with renewed binding capacity and high IgG selectivity.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
We report here a novel and simple cleaning method involving the use of polyethylene glycol (PEG8000) that renews matrix-binding capacity comparable to a new resin while retaining high selectivity for IgG. PMID: 19891968 [PubMed - as supplied by publisher] (Source: Journal of Immunological Methods)
Source: Journal of Immunological Methods - November 2, 2009 Category: Allergy & Immunology Authors: Owuor BO, Remarque EJ, Faber BW, Thomas AW Tags: J Immunol Methods Source Type: journals

A Novel Gamma-Fitting Statistical Method for Anti-Drug Antibody Assays to Establish Assay Cut-Points for Data with Non-Normal Distribution.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
In recent years there has been growing recognition of the impact of anti-drug or anti-therapeutic antibodies (ADAs, ATAs) on the pharmacokinetic and pharmacodynamic behavior of the drug, which ultimately affects drug exposure and activity. These anti-drug antibodies can also impact safety of the therapeutic by inducing a range of reactions from hypersensitivity to neutralization of the activity of an endogenous protein. Assessments of immunogenicity, therefore, are critically dependent on the bioanalytical method used to test samples, in which a positive versus negative reactivity is determined by a statistically deriv...
Source: Journal of Immunological Methods - November 2, 2009 Category: Allergy & Immunology Authors: Schlain B, Amaravadi L, Donley J, Wickramasekera A, Bennett D, Subramanyam M Tags: J Immunol Methods Source Type: journals

A Methodological Approach to Studies of Desensitization of the Formyl Peptide Receptor: Role of the read out system, reactive oxygen species and the specific agonist used to trigger neutrophils.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
In this study we show that desensitization patterns differ depending on what read out systems are used to follow neutrophil activity. When monitoring release of superoxide, neutrophils were readily desensitized against repeated stimulations with the prototypical agonist formylmethionyl-leucyl-phenylalanine (fMLF). In contrast, neutrophils were not desensitized for fMLF when cell activity was determined by intracellular calcium ([Ca(2+)](i)). The difference observed was dependent on inactivation of the agonist in one read out system but not in the other, and we suggest several different solutions to the problem. Agonist ina...
Source: Journal of Immunological Methods - November 2, 2009 Category: Allergy & Immunology Authors: Karlsson J, Bylund J, Movitz C, Björkman L, Forsman H, Dahlgren C Tags: J Immunol Methods Source Type: journals

High throughput screening for antibody induced complement-dependent cytotoxicity in early antibody discovery using homogeneous macroconfocal fluorescence imaging.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
In conclusion, we developed a novel high-throughput assay for the identification of therapeutic antibody lead candidates with optimal CDC characteristics from large antibody libraries. PMID: 19891971 [PubMed - as supplied by publisher] (Source: Journal of Immunological Methods)
Source: Journal of Immunological Methods - November 2, 2009 Category: Allergy & Immunology Authors: Gerritsen AF, Bosch M, de Weers M, van de Winkel JG, Parren PW Tags: J Immunol Methods Source Type: journals

Identification of a Sensitive Anti-Erythropoietin Receptor Monoclonal Antibody Allows Detection of Low Levels of EpoR in Cells.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Erythropoietin (Epo) binds and activates the Epo receptor (EpoR) on the surface of erythroid progenitor cells resulting in formation of erythrocytes. Recently, EpoR was reported to be expressed on nonerythroid cells suggesting a role for Epo outside of erythropoiesis. However those studies employed antibodies with questionable specificity and the significance of the observations are controversial. In order to accurately determine the expression of EpoR proteins in cells, we have generated a panel of novel anti-human EpoR monoclonal antibodies. One of these antibodies (A82) was particularly sensitive and it detected the...
Source: Journal of Immunological Methods - October 31, 2009 Category: Allergy & Immunology Authors: Elliott S, Busse L, McCaffery I, Rossi J, Sinclair A, Spahr C, Swift S, Begley CG Tags: J Immunol Methods Source Type: journals

Technical advancement in regulatory T cell isolation and characterization using CD127 expression in patients with malignant glioma treated with autologous dendritic cell vaccination.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
We have successfully treated over two hundred high-grade glioma (HGG) patients with immunotherapy consisting of vaccination with autologous dendritic cells (DCs) loaded with autologous tumour lysate. It has been documented that regulatory T cells (Treg) can counteract anti-tumour immune responses. Therefore, monitoring of Treg in these patients is essential. Up till now, Treg have been characterized based on the expression of the transcription factor Foxp3. Here, we validated IL-7 receptor alpha subunit (CD127)dim expression as a marker for human Treg within HGG patients, as a less laborious assay for routine use in tu...
Source: Journal of Immunological Methods - October 26, 2009 Category: Allergy & Immunology Authors: Ardon H, Verbinnen B, Maes W, Beez T, Van Gool S, De Vleeschouwer S Tags: J Immunol Methods Source Type: journals

Hybrid transgenic immune tolerant mouse model for assessing the breaking of B cell tolerance by human interferon beta.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
In this study, we used the original C57Bl/6 mouse model as well as the hybrid offspring from crossings of transgenic C57Bl/6 mice with wildtype FVB/N mice to study the immunogenicity of three commercial rhIFNbeta products, Rebif(R), Avonex(R) and Betaferon(R). As determined by ELISA, wildtype C57Bl/6 mice failed to form binding antibodies (BABs) against Rebif(R) and Avonex(R) formulated with human serum albumin. Because not all interferon beta products induce antibodies in wildtype C57Bl/6 mice, the transgenic C57Bl/6 mice cannot be used to study the breaking of tolerance by these products. However, the crossing of transge...
Source: Journal of Immunological Methods - October 23, 2009 Category: Allergy & Immunology Authors: van Beers MM, Sauerborn M, Gilli F, Hermeling S, Brinks V, Schellekens H, Jiskoot W Tags: J Immunol Methods Source Type: journals

Neurofilament ELISA validation.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
CONCLUSION: This multi-center validation study identified the lack of preparation of accurate and consistent protein standards as the main reason for a poor inter-laboratory CV. This issue is also relevant to other protein biomarkers based on this type of assay and will need to be solved in order to achieve an acceptable level of analytical accuracy. The raw data of this study is available online. PMID: 19857497 [PubMed - as supplied by publisher] (Source: Journal of Immunological Methods)
Source: Journal of Immunological Methods - October 23, 2009 Category: Allergy & Immunology Authors: Petzold A, Altintas A, Andreoni L, Bartos A, Berthele A, Blankenstein MA, Buee L, Castellazzi M, Cepok S, Comabella M, Constantinescu CS, Deisenhammer F, Deniz G, Erten G, Espiño M, Fainardi E, Franciotta D, Freedman M, Giedraitis V, Gilhus NE, Giova Tags: J Immunol Methods Source Type: journals

Comparison of the results obtained by ELISA and surface plasmon resonance for the determination of antibody affinity.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
PMID: 19854197 [PubMed - as supplied by publisher] (Source: Journal of Immunological Methods)
Source: Journal of Immunological Methods - October 22, 2009 Category: Allergy & Immunology Authors: Heinrich L, Tissot N, Hartmann DJ, Cohen R Tags: J Immunol Methods Source Type: journals

Analyses and comparisons of telomerase activity and telomere length in human T and B cells: Insights for epidemiology of telomere maintenance.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
We report the first quantitative data in humans examining both levels of telomerase activity and telomere length in four lymphocyte subpopulations from the same donors - CD4+, CD8+CD28+ and CD8+CD28- T cells and B cells, as well as total PBMCs - in a cohort of healthy women. We found that B cells had the highest telomerase activity and longest telomere length; CD4+ T cells had slightly higher telomerase activity than CD8+CD28+ T cells, and similar telomere length. Consistent with earlier reports that CD8+CD28- T cells are replicatively senescent cells, they had the lowest telomerase activity and shortest telomere length. I...
Source: Journal of Immunological Methods - October 14, 2009 Category: Allergy & Immunology Authors: Lin J, Epel E, Cheon J, Kroenke C, Sinclair E, Bigos M, Wolkowitz O, Mellon S, Blackburn E Tags: J Immunol Methods Source Type: journals

Azide and Tween-20 reduce binding to autoantibody epitopes of islet antigen-2; Implications for assay performance and reproducibility.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Autoantibodies to islet antigen 2 (IA-2A) are important markers for predicting diabetes in children and young adults. Harmonization of IA-2A assay measurement is essential if results from different laboratories are to be compared. We investigated whether sodium azide, a bacteriostatic agent added to some assays, could affect IA-2A binding and thereby contribute to differences in IA-2A measurement between laboratories. Addition of 0.1 % azide to assay buffer was found to reduce median IA-2A binding of 18 selected sera from IA-2A positive patients with type 1 diabetes and their relatives by 41 % (range, 78 to -33 %, p<...
Source: Journal of Immunological Methods - October 14, 2009 Category: Allergy & Immunology Authors: Williams AJ, Sommerville M, Rokni S, Bonifacio E, Yu L, Eisenbarth G, Akolkar B, Steffes M, Bingley PJ Tags: J Immunol Methods Source Type: journals

Measurement of Autoantibodies using Multiplex Methodology in patients with Systemic Lupus Erythematosus.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Autoantibodies are central to the diagnosis and assessment of systemic lupus erythematosus (SLE). A recent technique for the measurement of autoantibodies utilizes addressable laser bead immunoassay technology (BioPlex 2200) which permits the simultaneous detection of multiple autoantibodies and improved efficiency due to the shorter time to perform the assay and low volume of test samples and reagents. In the current study we have compared this technique to more traditional measures of autoantibody detection. The clinical and laboratory data and stored serum samples from the enrollment visit into a long-term lupus reg...
Source: Journal of Immunological Methods - October 13, 2009 Category: Allergy & Immunology Authors: Hanly JG, Thompson K, McCurdy G, Fougere L, Theriault C, Wilton K Tags: J Immunol Methods Source Type: journals

Detection of AMP-activated protein kinase in human sera by immuno-isoelectric focusing.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
AMPKalpha is a subunit of AMP-activated protein kinase (AMPK), a heterotrimeric enzyme that works as a fuel sensor activated in response to depletion of cellular ATP. AMPKalpha is considered as a master switch in regulating glucose and lipid metabolism. Determining its presence in patient sera may help to diagnose metabolic diseases. Using isoelectric focusing and Western blotting, we were able to detect AMPKalpha in human sera. Using specific antibodies, we showed that the AMPKalpha1 and alpha2 isoforms were apparently present in equal amounts in human sera. To characterize normal and abnormal AMPKalpha patterns, we u...
Source: Journal of Immunological Methods - October 6, 2009 Category: Allergy & Immunology Authors: Malvoisin E, Livrozet JM, El Hajji-Ridah I, Makloufi D, Vincent N Tags: J Immunol Methods Source Type: journals

Interferon-gamma release assay: A simple method for detection of varicella-zoster virus-specific cell-mediated immunity.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Herpes zoster is closely related to decreased varicella-zoster virus (VZV)-specific cell-mediated immunity. We validated a new assay for measuring VZV-specific immunity. We cultured the whole blood of healthy subjects with live attenuated VZV vaccine. Culture supernatants were harvested at 24-h intervals and assayed for interferon-gamma (IFN-gamma) by an enzyme-linked immunosorbent assay (ELISA). The 48-h culture was suitable for estimating IFN-gamma release. IFN-gamma production was stable after standing for at least 4h at room temperature. IFN-gamma production was observed in whole blood from subjects with recent VZV...
Source: Journal of Immunological Methods - October 6, 2009 Category: Allergy & Immunology Authors: Otani N, Baba K, Okuno T Tags: J Immunol Methods Source Type: journals

Improved serum-free culture conditions for the differentiation of human and murine fibrocytes.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
In this study, we describe enhanced serum-free conditions that support the rapid differentiation of human and murine fibrocytes. We compared the effect on fibrocyte differentiation of different anti-coagulants used when collecting blood, and for culturing cells, the effects of different commercial media formulations, the addition of a variety of supplements, cell density, conditioned medium, and glass and plastic substrates. We found that both heparin and EDTA were suitable anti-coagulants, but that blood treated with citrate-phosphate dextrose led to a reduced number of fibrocytes. Fibrocyte differentiation was enhanced w...
Source: Journal of Immunological Methods - October 6, 2009 Category: Allergy & Immunology Authors: Pilling D, Vakil V, Gomer RH Tags: J Immunol Methods Source Type: journals

Generation of macrophages from peripheral blood monocytes in the rhesus monkey.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
PMID: 19818793 [PubMed - as supplied by publisher] (Source: Journal of Immunological Methods)
Source: Journal of Immunological Methods - October 6, 2009 Category: Allergy & Immunology Authors: Rozner AE, Dambaeva SV, Drenzek JG, Durning M, Golos TG Tags: J Immunol Methods Source Type: journals

A novel method for isolating dendritic cells from human bronchoalveolar lavage fluid.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
CONCLUSION: Using a novel sorting strategy, functional mDCs can be isolated from BAL fluid, enabling detailed study in pulmonary disease. PMID: 19804781 [PubMed - as supplied by publisher] (Source: Journal of Immunological Methods)
Source: Journal of Immunological Methods - October 1, 2009 Category: Allergy & Immunology Authors: Ten Berge B, Muskens F, Kleinjan A, Hammad H, Hoogsteden HC, Lambrecht BN, Van den Blink B Tags: J Immunol Methods Source Type: journals

Two-photon laser scanning microscopy imaging of intact spinal cord and cerebral cortex reveals requirement for CXCR6 and neuroinflammation in immune cell infiltration of cortical injury sites.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
The mouse spinal cord is an important site for autoimmune and injury models. Skull thinning surgery provides a minimally invasive window for microscopy of the mouse cerebral cortex, but there are no parallel methods for the spinal cord. We introduce a novel, facile and inexpensive method for two-photon laser scanning microscopy of the intact spinal cord in the mouse by taking advantage of the naturally accessible intervertebral space. These are powerful methods when combined with gene-targeted mice in which endogenous immune cells are labeled with green fluorescent protein (GFP). We first demonstrate that generation of...
Source: Journal of Immunological Methods - September 30, 2009 Category: Allergy & Immunology Authors: Kim JV, Jiang N, Tadokoro CE, Liu L, Ransohoff RM, Lafaille JJ, Dustin ML Tags: J Immunol Methods Source Type: journals

Polymorphonuclear leukocytes isolated from umbilical cord blood as a useful research tool to study adherence to cell monolayers.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
In conclusion, our data suggest that umbilical cord blood is a suitable source of leukocytes to examine PMN adherence in the setting of inflammation in a variety of disease processes. PMID: 19800347 [PubMed - as supplied by publisher] (Source: Journal of Immunological Methods)
Source: Journal of Immunological Methods - September 29, 2009 Category: Allergy & Immunology Authors: Sharma J, Rastogi P, Creer MH, McHowat J Tags: J Immunol Methods Source Type: journals

Strategies of statistical image analysis of 2D immunoblots: The case of IgG response in experimental Taenia crassiceps cysticercosis.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
A procedure is described to measure the diversity and enrich the meaning and usefulness of the information contained in 2D-immunoblot images of the reaction between a complex mixture of parasite antigens with the complex set of antibodies usually present in the sera of infected individual hosts. The procedure and results are illustrated by the experimental infection of 30 mice (three strains, both sexes, 5 mice in each strain x sex combination) with Taenia crassiceps cysticerci, thirty days after challenge. The exercise revealed a significant positive correlation of parasite loads with the hosts IgG response, in associ...
Source: Journal of Immunological Methods - September 29, 2009 Category: Allergy & Immunology Authors: Ostoa-Saloma P, Esquivel-Velázquez M, Ostoa-Jacobo P, Larralde C Tags: J Immunol Methods Source Type: journals

Neutralizing antibodies against IFN beta in patients with multiple sclerosis: A comparative study of two cytopathic effect tests (CPE) for their detection.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
PMID: 19786034 [PubMed - as supplied by publisher] (Source: Journal of Immunological Methods)
Source: Journal of Immunological Methods - September 24, 2009 Category: Allergy & Immunology Authors: Oliver B, Orpez T, Mayorga C, Pinto-Medel MJ, Leyva L, López-Gómez C, Marín C, Luque G, Ortega-Pinazo J, Fernández O Tags: J Immunol Methods Source Type: journals

Comparison of three assays for the detection of GAD65Ab-specific anti-idiotypic antibodies.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
In this study we evaluated two novel detection assays for GAD65Ab-specific anti-Id. The biotin/streptavidin based absorption assay utilizes the strong interaction of biotin and streptavidin to prevent possible leakage of the immobilized antibody. Moreover, this assay format allows to identify the origin of the detected GAD65Ab. The ECL-based assay allows the direct detection of anti-Id independent of the presence of GAD65Ab. We analyzed new-onset type 1 diabetes patients (n=133) and matched healthy controls (n=178) for the presence of GAD65Ab-specific anti-Id using both new detection assays and the original absorption assa...
Source: Journal of Immunological Methods - September 21, 2009 Category: Allergy & Immunology Authors: Oak S, Radtke J, Landin-Olsson M, Törn C, Lernmark A, Hampe CS Tags: J Immunol Methods Source Type: journals

Methods for comprehensive identification of membrane proteins recognized by a large number of monoclonal antibodies.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
In order to isolate monoclonal antibodies (mAbs) that bind to tumor-associated antigens (Ags) we developed the following strategy. Using the phage-display Ab library we isolated a large number of mAbs that bind to the surface of human tumor cells. The mAbs were individually screened by immunostaining, and clones that preferentially and strongly stained the malignant cells were chosen. Thereafter, the Ags recognized by the mAbs were identified. For identification of the Ags by MS candidate molecules had to be purified either by immunoprecipitation or by affinity chromatography. We isolated several hundred mAbs that show...
Source: Journal of Immunological Methods - September 16, 2009 Category: Allergy & Immunology Authors: Kurosawa G, Sumitomo M, Akahori Y, Matsuda K, Muramatsu C, Takasaki A, Iba Y, Eguchi K, Tanaka M, Suzuki K, Morita M, Sato N, Sugiura M, Sugioka A, Hayashi N, Kurosawa Y Tags: J Immunol Methods Source Type: journals

Development and use of a novel in vitro assay for testing of diphtheria toxoid in combination vaccines.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Testing of diphtheria vaccines for routine lot release relies heavily on the use of in vivo potency assays. However, consistency of production is also recognised as an important feature of vaccine manufacture, and in vitro assays are superior to in vivo assays for providing this information. In adsorbed vaccines, antigen and adjuvant are the major components contributing to immunogenicity and are therefore critical factors to be evaluated as part of consistency testing. Here we describe a simple and sensitive Enzyme Linked Immunosorbent Assay (ELISA) which has been developed to quantify diphtheria toxoid antigen in com...
Source: Journal of Immunological Methods - September 13, 2009 Category: Allergy & Immunology Authors: Coombes L, Stickings P, Tierney R, Rigsby P, Sesardic D Tags: J Immunol Methods Source Type: journals

Validity of multiplex-based assays for cytokine measurements in serum and plasma from "non-diseased" subjects: Comparison with ELISA.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
In conclusion, multiplex-based assays can give an accurate estimate of the relative risk in large-scale epidemiological studies but only for cytokines that are present in relatively large concentrations (ng/mL). PMID: 19748508 [PubMed - as supplied by publisher] (Source: Journal of Immunological Methods)
Source: Journal of Immunological Methods - September 10, 2009 Category: Allergy & Immunology Authors: Dossus L, Becker S, Achaintre D, Kaaks R, Rinaldi S Tags: J Immunol Methods Source Type: journals

A single-step procedure of recombinant library construction for the selection of efficiently produced llama VH binders directed against cancer markers.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Heavy chain antibodies are naturally occurring in camelidae (camels and llamas). Their variable domain (VHH) can be efficiently produced as a recombinant protein in E. coli with a large range of applications in the fields of diagnostics and immunotherapy. Standard cloning approach involves resolution of VHH from the heavy chain variable domain of conventional antibodies (VH) by a nested PCR amplification followed by a phage display based selection. Present work illustrates that in contrast to usual finding, specific, good affinity and efficiently expressed VH domain of conventional antibodies can be selected from the c...
Source: Journal of Immunological Methods - September 6, 2009 Category: Allergy & Immunology Authors: Kastelic D, Frković-Grazio S, Baty D, Truan G, Komel R, Pompon D Tags: J Immunol Methods Source Type: journals

Quantification of IFN-gamma produced by human purified NK cells following tumor cell stimulation: Comparison of three IFN-gamma assays.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
In this study, we assessed the relationship between stimulation of human purified resting peripheral blood NK cells with one (tumor cell or IFN-alpha) and two (tumor cell+IFN-alpha) signals by measuring IFN-gamma using three different assays. We performed the enzyme-linked immunosorbent assay (ELISA), the enzyme-linked immunospot (ELISPOT) assay and intracellular cytokine staining (ICS) assay in parallel per donor and determined whether there was a correlation between these assays. Our results show that two-signal stimulation of human resting NK cells induces significantly more IFN-gamma as compared to one-signal stimulati...
Source: Journal of Immunological Methods - September 4, 2009 Category: Allergy & Immunology Authors: Lion E, Smits EL, Berneman ZN, Van Tendeloo VF Tags: J Immunol Methods Source Type: journals

Rapid detection of antibodies in sera using multiplexed self-assembling bead arrays.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Rapid detection of antibody immunity in serum or plasma, whether to pathogenic antigens, tumor antigens, or autoimmune antigens, is critical for diagnosis, monitoring, and biomarker assessment of the immune response. Individual or multiplexed ELISAs that use purified recombinant proteins are dependent on a priori protein purification, a labor-intensive process that may take months to obtain proteins of sufficient purity and stability for serologic assays. We developed a programmable multiplexed immunoassay for the rapid monitoring of humoral immunity using the Luminex suspension bead array platform. In this approach, e...
Source: Journal of Immunological Methods - September 2, 2009 Category: Allergy & Immunology Authors: Wong J, Sibani S, Lokko NN, Labaer J, Anderson KS Tags: J Immunol Methods Source Type: journals

Simultaneous detection of ERK-, p38-, and JNK-MAPK phosphorylation in human adipose-derived stem cells using the Cytometric Bead Array technology.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Despite expanded research in stem cell biology, little is known about the mechanisms underlying migration, growth, and differentiation of adipose-derived adult mesenchymal stem cells (ASC). The simultaneous measurement of intracellular pathways opens new avenues to gain further insights in these processes. We used the Cytometric Bead Array (CBA) Flex Set technology to simultaneously analyze protein phosphorylation after stimulation of ASC and compared the results with data generated by corresponding Western blots. Signal transduction of ASC was stimulated by epidermal growth factor (EGF) and analyzed by determining pho...
Source: Journal of Immunological Methods - September 2, 2009 Category: Allergy & Immunology Authors: Schubert R, Geiger H, Zielen S, Baer PC Tags: J Immunol Methods Source Type: journals

Analytical validation of a highly sensitive microparticle-based immunoassay for the quantitation of IL-13 in human serum using the Erenna(R) immunoassay system.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
IL-13 is a Th2 cytokine that has been shown to be an important mediator of airway inflammation contributing to asthma lesions. Given its proposed role in asthma, measurements of this cytokine in serum may provide insights into disease mechanisms, progression and pharmacodynamic effects of IL-13 targeted therapeutics. However, current commercially available ELISA immunoassays are frequently unable to detect baseline concentrations of IL-13 in serum from healthy individuals, which are below the limit of detection. Here we describe the use of the novel microparticle-based Erenna((R)) IL-13 human immunoassay (Singulex, Inc...
Source: Journal of Immunological Methods - September 1, 2009 Category: Allergy & Immunology Authors: Ledger KS, Agee SJ, Kasaian MT, Forlow SB, Durn BL, Minyard J, Lu QA, Todd J, Vesterqvist O, Burczynski ME Tags: J Immunol Methods Source Type: journals

ELISPOT and functional T cell analyses using HLA mono-specific target cells.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Simple T cell assays specific for any chosen HLA class I or class II/peptide combination, are of enormous value in cancer immunotherapy, clinical trials, vaccine and infectious disease research. The reliable measurement of T cell activity can be difficult due to the presence of other alleles on target cells, particularly for the non-HLA-A2 alleles, and the varying baseline characteristics of the different APCs employed. In the absence of pulsing with HLA-A2 restricted peptides, T2 cells are functionally HLA class I and II negative. By coating these cells with recombinant HLA peptide complexes, HLA mono-specific cells a...
Source: Journal of Immunological Methods - August 30, 2009 Category: Allergy & Immunology Authors: Horlock C, Stott B, Dyson J, Ogg G, McPherson T, Jones L, Sewell A, Wooldridge L, Cole D, Stebbing J, Savage P Tags: J Immunol Methods Source Type: journals

Novel method for quantitative ANA measurement using near-infrared imaging.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
In this study, we describe a simple quantitative method to detect ANA which combines IIF on HEp-2 coated slides with analysis using a near-infrared imaging (NII) system. Using NII to determine ANA titer, 86.5% (32 of 37) of the titers for human patient samples were within 2 dilutions of those determined by IIF, which is the acceptable range for proficiency testing. Combining an initial screening for nuclear staining using microscopy with titration by NII resulted in 97.3% (36 of 37) of the titers detected to be within two dilutions of those determined by IIF. The NII method for quantitative ANA measurements using serum fro...
Source: Journal of Immunological Methods - August 28, 2009 Category: Allergy & Immunology Authors: Peterson LK, Wells D, Shaw L, Velez MG, Harbeck R, Dragone LL Tags: J Immunol Methods Source Type: journals

Cloning and expression of murine Ig genes from single B cells.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
We have established a highly efficient 96-well format based strategy to characterize the expressed murine antibody repertoire by combining immunoglobulin (Ig) gene cloning with antibody expression and reactivity profiling at the single cell level. Individual mouse B lineage cells are isolated based on defined surface marker expression patterns by fluorescence-activated cell sorting (FACS) and corresponding full-length Ig heavy (H) and Ig light (L) chain variable (V) region gene transcripts are amplified by RT-PCR. Cloning of the amplified products into eukaryotic expression vectors enables the in vitro production of mo...
Source: Journal of Immunological Methods - August 25, 2009 Category: Allergy & Immunology Authors: Tiller T, Busse CE, Wardemann H Tags: J Immunol Methods Source Type: journals

Some lessons from the systematic production and structural analysis of soluble alphabeta T-cell receptors.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
T-cell receptors (TCRs) are membrane proteins which recognize antigens with high specificity forming the basis of the cellular immune response. The study of these receptors has been limited by the challenges in expressing sufficient quantities of stable soluble protein. Here we report our systematic approach for generating soluble, alphabetaTCRs, for X-ray crystallographic studies. By using small-scale expression screens, novel standardized quality control mechanisms and crystallization and imaging robots we were able to add significantly to the current TCR structural database. Our success in crystallizing both isolate...
Source: Journal of Immunological Methods - August 24, 2009 Category: Allergy & Immunology Authors: van Boxel GI, Stewart-Jones G, Holmes S, Sainsbury S, Shepherd D, Gillespie GM, Harlos K, Stuart DI, Owens R, Jones EY Tags: J Immunol Methods Source Type: journals

Expression of blood coagulation factors on monocytes after exposure to TNF treated endothelium in a novel whole blood model of arterial flow.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Activated blood monocytes are a major source of Tissue Factor (TF), the principal initiator of blood coagulation. TF can be shed from the monocyte surface in association with microparticles (MPs) and increased numbers of circulating MPs are indicative of poor clinical outcome in a number of inflammatory disorders, including atherosclerosis. The mechanisms coupling inflammation with aberrant TF production/activity remain obscure but the protease-activated receptor (PAR) family has been implicated. We have previously shown (i) that freshly isolated human monocytes express low levels of cell surface PAR-2, (ii) that cell ...
Source: Journal of Immunological Methods - August 19, 2009 Category: Allergy & Immunology Authors: Macey MG, Wolf SI, Wheeler-Jones CP, Lawson C Tags: J Immunol Methods Source Type: journals

Differences in usability of rabbit IgG and chicken IgY after clean-up and impact on gold labelling properties.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
For the application of antibodies in rapid test systems such as Lateral Flow Devices (LFD) antibodies have to be coupled to coloured particles for immediate readability of the test system. In this work colloidal gold was selected for conjugation to the antibodies. Polyclonal rabbit antibodies were chosen for the development of Lateral Flow Devices for the detection of bovine alpha-casein. For antibody comparison chicken egg yolk IgY and sheep IgG were additionally used. Rabbit and chicken antibodies were purified from rabbit sera and egg yolk using affinity chromatography and alternatively ammonium sulphate precipitati...
Source: Journal of Immunological Methods - August 19, 2009 Category: Allergy & Immunology Authors: Rudolf J, Führer M, Galler B, Ansari P, Hasenhindl C, Baumgartner S Tags: J Immunol Methods Source Type: journals

A novel measurement method for activation of the lectin complement pathway via both mannose-binding lectin (MBL) and L-ficolin.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
In conclusion, we propose a novel method using GN5-DPPE for investigating the MBL- and L-ficolin-dependent lectin pathway and anticipate that this method will be useful in innate immunity and clinical research. PMID: 19699205 [PubMed - as supplied by publisher] (Source: Journal of Immunological Methods)
Source: Journal of Immunological Methods - August 18, 2009 Category: Allergy & Immunology Authors: Inoshita H, Matsushita M, Koide S, Kusaba G, Ishii M, Onda K, Gi MJ, Nakata M, Ohsawa I, Horikoshi S, Ohi H, Tomino Y Tags: J Immunol Methods Source Type: journals

Large scale expansion of Vgamma9Vdelta2 T lymphocytes from human peripheral blood mononuclear cells after a positive selection using MACS "TCR gamma/delta+ T cell isolation kit".Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
In this study, we describe a straightforward protocol to amplify pure populations of gamma9delta2 T cells which could be useful in fundamental research or in the development of a new generation of gammadelta cell therapy protocol. PMID: 19465023 [PubMed - indexed for MEDLINE] (Source: Journal of Immunological Methods)
Source: Journal of Immunological Methods - August 1, 2009 Category: Allergy & Immunology Authors: Salot S, Bercegeay S, Dreno B, Saïagh S, Scaglione V, Bonnafous C, Sicard H Tags: J Immunol Methods Source Type: journals

ELDA: extreme limiting dilution analysis for comparing depleted and enriched populations in stem cell and other assays.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
ELDA is a software application for limiting dilution analysis (LDA), with particular attention to the needs of stem cell assays. It is the first limiting dilution analysis software to provide meaningful confidence intervals for all LDA data sets, including those with 0% or 100% responses. Other features include a test of the adequacy of the single-hit hypothesis, tests for frequency differences between multiple data sets, and the ability to take advantage of cases where the number of cells in the sample is counted exactly. A webtool at http://bioinf.wehi.edu.au/software/elda/ provides an easy user interface. PMID: ...
Source: Journal of Immunological Methods - August 1, 2009 Category: Allergy & Immunology Authors: Hu Y, Smyth GK Tags: J Immunol Methods Source Type: journals

Review on enzyme-linked immunosorbent assays for sulfonamide residues in edible animal products.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
The current status of enzyme-linked immunosorbent assays (ELISAs) for sulfonamides in edible animal products is reviewed. The attention was focused on the design and synthesis of haptens, conjugation to carrier protein, production of antibody, application of homologous and heterologous systems, as well as the molecular modeling of the haptens and sulfonamides. Researches have shown that sulfonamides seem to be particularly resistant to attempts to produce broad specificity antibodies. By summarizing the available research on sulfonamide ELISAs, it is hoped that it can be considered as a basis for further investigation ...
Source: Journal of Immunological Methods - July 26, 2009 Category: Allergy & Immunology Authors: Zhang H, Wang S Tags: J Immunol Methods Source Type: journals

Preparation and validation of radio iodinated recombinant human il-10 for the measurement of natural human antibodies against IL-10.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Radio iodinated recombinant human IL-10 was prepared and validated for the measurement of natural human anti-IL-10 antibodies. Iodination of IL-10 was accomplished by means of the chloramine-T method. The crude tracer was purified by size chromatography as homodimeric IL-10 with a specific activity of 75 cpm/pg. Validation of the tracer confirmed preserved antibody epitopes and receptor binding ability. A robust Radio Immuno Assay (RIA) was developed and validated to detect natural human anti-IL-10 antibodies based on the formation of (125)I-labeled IL-10-IgG complexes in solution and separation of the complexes by chr...
Source: Journal of Immunological Methods - July 22, 2009 Category: Allergy & Immunology Authors: Rieper CD, Galle P, Svenson M, Pedersen BK, Hansen MB Tags: J Immunol Methods Source Type: journals

Molecular characterization of hybridoma subclones spontaneously switching at high frequencies in vitro.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
The hybridoma technology allows the production of large quantities of specific antibodies of a single isotype. Since different isotypes have special effector functions and are distributed distinctively throughout the body, it is often useful to have a library of switch variants from the original monoclonal antibody. We have shown previously that forced expression of activation induced cytidine deaminase (AID) in hybridomas increased their very low frequency of class switch recombination (CSR) in vitro only ~7-13 fold. Since we had previously identified rare hybridoma subclones that spontaneously switched at more than 1...
Source: Journal of Immunological Methods - July 16, 2009 Category: Allergy & Immunology Authors: Iglesias-Ussel MD, Zavadil J, Scharff MD Tags: J Immunol Methods Source Type: journals

Flow cytometric calcium flux assay: Evaluation of cytoplasmic calcium kinetics in whole blood leukocytes.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
In conclusion, the present method is a simple, fast and easy tool to analyse in vitro [Ca(2+)](i) changes over time in leukocytes under physiologically relevant conditions, without the need for their isolation or the lysis of erythrocytes. The whole blood approach allows a continuous interaction between the different leukocyte subpopulations and other blood components and a minimum of preparative manipulations avoids artefactual activation of the cells. A distinction can be made between Ca(2+) release from the intracellular stores and the entry of Ca(2+) from outside the cell. The approach allows to evaluate the effect of ...
Source: Journal of Immunological Methods - July 15, 2009 Category: Allergy & Immunology Authors: Schepers E, Glorieux G, Dhondt A, Leybaert L, Vanholder R Tags: J Immunol Methods Source Type: journals