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Journal of Immunological Methods

Melissa Hladek, Sarah L Szanton, Young-Eun Cho, Chen Lai, Caroline Sacko, Laken Roberts, Jessica Gill
BACKGROUND/OBJECTIVES: Current measures of cytokines involve urine, blood or saliva which have drawbacks including circadian rhythm variations and complicated collection methods. Sweat has been used to measure cytokines in young and middle-aged adults, but not older adults. We sought to determine the feasibility of using sweat to measure cytokines in older adults compared to younger adults. DESIGN: Two visit cross-sectional pilot study stratified by age group. SETTING: Independent living facility and Johns Hopkins University both in Maryland...
November 8, 2017: Journal of Immunological Methods
Nicole Hartwig Trier, Bettina Eide Holm, Julie Heiden, Ole Slot, Henning Locht, Bente Jensen, Hanne Lindegaard, Anders Svendsen, Christoffer Tandrup Nielsen, Søren Jacobsen, Elke Theander, Gunnar Houen
Rheumatoid arthritis (RA) is an autoimmune disease of unknown etiology. A characteristic feature of RA is the presence of anti-citrullinated protein antibodies (ACPA). Since ACPAs are highly specific for RA and are often present before the onset of RA symptoms, they have become valuable diagnostic and prognostic. As a result, several assays for detection of ACPAs exist, which vary in sensitivity and specificity. In this study, we analyzed the reactivity of RA sera to selected peptides by solid-phase immunoassays in order to develop an ACPA assay with improved sensitivity and specificity...
November 8, 2017: Journal of Immunological Methods
Muir Russell, Abraham Mellkvist-Roos, John Mo, Rabia Hidi
We describe a simple and robust tube-based ex vivo whole blood stimulation procedure suitable for use in clinical laboratories by multiple operators on repeated occasions to study cytokine production in phase 1 human trials of investigational medicines, developed by the authors specifically to study IL-17A expression in man. The stimulation procedure is further proposed as a useful tool for biomarker assay development and validation, for example to prepare quality control samples without reliance on recombinant materials for spiking...
November 7, 2017: Journal of Immunological Methods
Alexander P Sung, Jennifer J-J Tang, Michael J Guglielmo, Doug Redelman, Julie Smith-Gagen, Lucinda Bateman, Dorothy Hudig
Natural killer (NK) lymphocyte ADCC supports anti-viral protection and monoclonal antibody (mAb) anti-tumor therapies. To predict in vivo ADCC therapeutic responses of different individuals, measurement of both ADCC cellular lytic capacity and their NK cellular receptor recognition of antibodies on 'target' cells are needed, using clinically available amounts of blood. Twenty ml of blood provides sufficient peripheral blood mononuclear cells (PBMCs) for the new assay for lytic capacity described here and for an antibody EC50 assay for Fc-receptor recognition...
November 4, 2017: Journal of Immunological Methods
Chong Sun, Jianmin Li, Bin Wang, Junjie Shangguan, Matteo Figini, Na Shang, Liang Pan, Zhuoli Zhang
PURPOSE: To test the hypotheses that pathological biomarkers including CD34 positive endothelial cell and microvessel density (MVD) of the primary breast tumor can be used to predict the probability of occurrence for bone metastases and provide information for appropriate therapeutic strategies at an early stage. METHODS: Three groups of CD34 immunohistochemical stained slices (n=60) were acquired from surgical specimens of sixty patients including non-metastasis (group 1), only lymph node metastasis (group 2), and bone metastasis (group 3)...
October 21, 2017: Journal of Immunological Methods
Liwei He, Jin Su, Marin Ming, Lidice Bernardo, Tricia Chen, Lucy Gisonni-Lex, Beata Gajewska
We have developed an accurate, precise and stability-indicating flow cytometry (FC) based assay to directly measure antigenicity of H4 protein (also known as HyVac4) in a vaccine formulation of H4-IC31, without desorbing the H4 protein from the IC31 adjuvant. This method involves immuno-staining of H4-IC31 complex with anti-H4 monoclonal antibodies (mAbs) followed by FC analysis. The assay is not only able to consistently measure H4 antigenicity levels in H4-IC31 stored under normal condition at 2-8°C, but also able to detect changes in H4 antigenicity after H4-IC31 undergoes heat stress or freeze-thawing...
October 19, 2017: Journal of Immunological Methods
Ingrid Elisia, Han Bee Pae, Vivian Lam, Rachel Cederberg, Elyse Hofs, Gerald Krystal
The RAW264.7 mouse macrophage cell line is used extensively to carry out in vitro screens for immunomodulators. Compounds that are effective at reducing the expression of pro-inflammatory cytokines or nitric oxide (NO) from lipopolysaccharide (LPS)-stimulated RAW264.7 cells are often considered candidate anti-inflammatory agents for humans. There is, however, very little data on the reliability of this screen to identify bona fide human immunomodulators. We compared the efficacy of 37 purported immunomodulators to modulate LPS or E...
October 16, 2017: Journal of Immunological Methods
Limor Cohen, Liangxia Xie, Mark E Xylas, David R Walt
Rats are used as animal models for many human diseases. Cytokines can serve as biomarkers indicative of these diseases or disease states. Techniques for measuring cytokine expression levels often do not provide the sensitivity needed to measure these biomarkers in biological fluids because the concentrations of many cytokines are below the detection limits of conventional methods. In this paper, we present ultra-sensitive digital immunoassays using Single Molecule Arrays (Simoa) for seven rat cytokines: TNF-α, IL-10, IL-17F, GM-CSF, IFN-γ, IL-4, and IL-1α...
October 14, 2017: Journal of Immunological Methods
Maohua Li, Hongzhen Li, Kai Gao, Muyang Wang, Wenqi An, Yaran Zhu, Li Ding, Lan Wang, Jingliang Gu, Conglin Zuo, Le Sun
It has been reported that 90% of the anti-drug antibody (ADA) to Adalimumab in human patients bound to the TNF-binding area, resulted in the annual loss of responses to Adalimumab up to 24%. It is of urgency to develop a cost-effective and easy-to-use ADA diagnostic kit for diagnosis of potential drug-resistance in patients treated with Adalimumab in clinic hospitals to avoid the tremendous economic and human costs to patients and health-care providers. In this study, we reported the generations of mouse monoclonal and monkey polyclonal antibodies against Adalimumab as assay standards and positive quality controls respectively...
October 13, 2017: Journal of Immunological Methods
Angela Ceribelli, Natasa Isailovic, Maria De Santis, Elena Generali, Minoru Satoh, Carlo Selmi
OBJECTIVE: To describe a new immunoprecipitation pattern identified in Italian patients affected by systemic sclerosis (SSc), corresponding to the pyruvate dehydrogenase antigen complex recognized by anti-mitochondrial antibodies (AMA) in primary biliary cholangitis (PBC). METHODS: Autoantibodies in sera from 85 patients with SSc were tested by protein- and RNA-immunoprecipitation. Immunoprecipitation-Western blot was used to determine the identified proteins, and medical records re-evaluated for liver function tests and PBC...
October 4, 2017: Journal of Immunological Methods
M Fehlings, S Chakarov, Y Simoni, B Shivshankar, F Ginhoux, E W Newell
Antigen-specific T cells play a crucial role for the host protective immunity against viruses and other diseases. The combination of mass cytometry together with a combinatorial multiplex tetramer staining has successfully been applied for probing and characterization of multiple antigen-specific CD8(+) T cells in human blood samples. The present study shows that this approach can also be used to rapidly identify the magnitude of influenza-specific CD8(+) T cell peptide dominance across lymph nodes and lungs in a murine model of a highly pathological influenza infection...
September 30, 2017: Journal of Immunological Methods
Wenfang S Wu, Liang Zhu, Saurabh Patil, Karthiga Gokul, Sean Reilly, Joyce Chan, Poornima Tekumalla, Vivek Vishnudas, Michael A Kiebish, Rangaprasad Sarangarajan, Viatcheslav R Akmaev, Mark D Kellogg, Niven R Narain
Coiled-Coil Domain Containing 47 (CCDC47) is an endoplasmic reticulum (ER) transmembrane protein involved in calcium signaling through utilization of its calcium binding-acidic luminal domain. CCDC47 also interacts with ERAD (endoplasmic reticulum-associated degradation) complex and is involved in ER stress relief. In this report, we developed human CCDC47 monoclonal antibodies and a sandwich immunoassay for CCDC47 measurement in biological matrices. Specificity of developed antibodies were confirmed by immunoblot and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of immunoprecipitated cell lysates...
September 30, 2017: Journal of Immunological Methods
Hiroko Shibata, Kazuko Nishimura, Chizuru Miyama, Minoru Tada, Takuo Suzuki, Yoshiro Saito, Akiko Ishii-Watabe
Development of an appropriate assay to detect anti-drug antibody (ADA) is important for assessing immunogenicity to therapeutic protein products. However, characterizing ADA assay methods is difficult because human ADA as a reference standard is not available in most cases. We compared the analytical performance of three ligand-binding assay methods for ADA, namely, surface plasmon resonance (SPR), electrochemiluminescence (ECL), and biolayer interferometry (BLI) methods, by using the anti-erythropoietin (EPO) monoclonal antibody reference panel developed by the World Health Organization (WHO) in 2015...
September 29, 2017: Journal of Immunological Methods
Elena Blanco, Martin Perez-Andres, Luzalba Sanoja-Flores, Marjolein Wentink, Ondrej Pelak, Marta Martín-Ayuso, Georgiana Grigore, Juan Torres-Canizales, Eduardo López-Granados, Tomas Kalina, Mirjam van der Burg, Sonia Arriba-Méndez, Santiago Santa Cruz, Noemí Puig, Jacques J M van Dongen, Alberto Orfao
The clinical value of assessing immunoglobulin (Ig)G and IgA subclasses in addition to the isotypes of soluble Igs in serum has been well established. >20years ago, the International Union of Immunological Societies and the World Health Organization performed collaborative studies in order to validate antibody (Ab) clones for the detection of IgG and IgA subclasses for a broad range of laboratory assays, except for flow cytometry. Here we analyzed the performance of commercially available Ab clones to detect IgG and IgA subclasses in memory B-cells and plasma cells (PCs) by flow cytometry...
September 28, 2017: Journal of Immunological Methods
G Yadirgi, P Stickings, S Rajagopal, Y Liu, D Sesardic
Botulinum toxin type A is a causative agent of human botulism. Due to high toxicity and ease of production it is classified by the Centres for Disease Control and Prevention as a category A bioterrorism agent. The same serotype, BoNT/A, is also the most widely used in pharmaceutical preparations for treatment of a diverse range of neuromuscular disorders. Traditionally, animals are used to confirm the presence and activity of toxin and to establish neutralizing capabilities of countermeasures in toxin neutralization tests...
September 21, 2017: Journal of Immunological Methods
Jessica A White, Candace Haghighi, Johanna Brunner, Marcus Estrada, Manjari Lal, Dexiang Chen
Double mutant heat-labile toxin (dmLT) is a promising adjuvant for oral vaccine administration. The aims of our study were to develop sensitive methods to detect low concentrations of dmLT and to use the assays in preformulation studies to determine whether dmLT remains stable under conditions encountered by an oral vaccine. We developed a sandwich ELISA specific for intact dmLT and a sensitive SDS-PAGE densitometry method, and tested stability of dmLT in glass and plastic containers, in saliva, at the pH of stomach fluid, and in high-osmolarity buffers...
September 20, 2017: Journal of Immunological Methods
Sarita Das, T Dileepan, D R Johnson, E L Kaplan, P Patrick Cleary
Among the four known Streptococcal nucleases comprising of DNase A, B, C and D; DNase B is the most common, and determination of the levels of antibody to DNase B (ADB) is often used to confirm a clinical diagnosis of Streptococcus pyogenes/group A Streptococcal (GAS) infection. The commonly used assays for antibodies that neutralize DNase B or streptolysin O activity use partially purified antigens that often fail to detect antibody changes subsequent to culture documented infections. Therefore, an enzyme-linked immunosorbent assay (ELISA) was developed employing his-tagged recombinant DNase B as plate antigen for comparison to the commonly used DNA methyl green micromethod (DMGM)...
September 20, 2017: Journal of Immunological Methods
Sanjukta Chatterjee, Laxmikant Vashishta, Vinit S Waichale, Vivek G Nayak, Ramakrishnan Melarkode, Charles M Donnelly, Patrick T Vallano, Narendra Chirmule, Nilanjan Sengupta
Recombinant biopharmaceuticals can induce generation of anti-drug antibodies, which could potentially neutralize therapeutic drug activity. In this report, we describe development and validation of a cell-based assay for detection of neutralizing antibodies (Nab) against insulin and insulin analogues. In order to achieve clinically meaningful sensitivity the method used an early signalling event, insulin induced insulin receptor phosphorylation as the endpoint. Percentage insulin receptor phosphorylation in cell lysates was measured using ECL based ELISA...
September 18, 2017: Journal of Immunological Methods
Umberto Basile, Francesca Gulli, Laura Gragnani, Cecilia Napodano, Krizia Pocino, Gian Ludovico Rapaccini, Michele Mussap, Anna Linda Zignego
The production of antibodies is accompanied by a slight excess of synthesis of κ and λ immunoglobulin light chains; small amounts of them are released in the peripheral blood and can also be found in various body fluids, such as synovial fluid, cerebrospinal fluid, urine and saliva. They are rapidly filtered by the glomerulus and >99% are reabsorbed from the cells of the proximal convoluted tubule, making them present in the urine in only trace amounts. The production of an excess of protein without a reason or a specific function in a biological system is rare...
September 18, 2017: Journal of Immunological Methods
R Madelaine Paredes, Douglas K Tadaki, Amanda Sooter, Fabia Gamboni, Forest Sheppard
Immunophenotyping of whole blood (WB) by flow cytometry (FC) is used clinically to assess a patient's immune status and also in biomedical research. Current protocols recommend storage of immunolabeled samples at 4°C with FC analysis to be completed within seven days. This data acquisition window can be extended to up to one year post-labeling, but this requires cryopreservation of the samples at ultra-low temperatures (≤-80°C or in liquid nitrogen). In this study we optimized a standardized cryopreservation protocol to enable preservation of immunolabeled, human WB samples at -20°C for FC and tested its effectiveness after 0, 5, 15 or 30days...
September 18, 2017: Journal of Immunological Methods
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