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Journal of Immunological Methods

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https://www.readbyqxmd.com/read/28434982/novel-monoclonal-antibodies-against-stx1d-and-1e-and-their-use-for-improving-immunoassays
#1
Xiaohua He, Stephanie Patfield, Reuven Rasooly, Daniela Mavrici
Shiga toxins (Stxs) are major causative agents for bloody diarrhea and hemolytic uremic syndrome, a life-threatening disease in humans. No effective treatment is available. Early detection of Stxs in clinical samples is critical for disease management. As bacteria evolve, new Stxs are produced; therefore, methods used to identify them need to be improved as well. In this study, new monoclonal antibodies (mAbs) against Stx1d and 1e were developed and used to improve a commercial Stx1 kit. Incorporation of the new mAbs into the Abraxis Stx1 kit not only increased the assay sensitivity to Stx1d, but the assay was conferred the ability to detect Stx1e, a newly identified subtype of Stx1 produced by an atypical Stx-producing bacterial strain, Enterobacter cloacae M12X01451, isolated from a clinical specimen...
April 20, 2017: Journal of Immunological Methods
https://www.readbyqxmd.com/read/28434981/protein-transport-inhibitors-downregulate-the-expression-of-lag-3-on-regulatory-t-cells
#2
Sara Anvari, Andrea Grimbergen, Carla M Davis, George Makedonas
Modern immunologic studies demand increasing complexity because of a need to improve our understanding of the relationship between a cell's phenotype and its function. Regulatory T cells have been defined by a narrow set of phenotypic markers, however their actual functional capacity has not been determined at the single-cell level. Although the lymphocyte activation gene 3 (LAG-3; CD223) is a key marker for the identification of exhausted T cells, it may be useful also in resolving Treg subpopulations by indicating distinct functional breadths...
April 20, 2017: Journal of Immunological Methods
https://www.readbyqxmd.com/read/28434980/thaw-and-use-target-cells-pre-labeled-with-calcein-am-for-antibody-dependent-cell-mediated-cytotoxicity-assays
#3
Shan Chung, Van Nguyen, Yuwen Linda Lin, Lynn Kamen, An Song
In vitro antibody-dependent cell-mediated cytotoxicity (ADCC) assays are routinely performed to support the research and development of therapeutic antibodies. In ADCC assays, target cells bound by the antibodies are lysed by activated effector cells following interactions between the Fc region of the bound antibody and Fcγ receptors on effector cells. Target cell lysis is typically measured by quantification of released endogenous enzymes, e.g., lactate dehydrogenase, or measurement of released exogenous labels, e...
April 20, 2017: Journal of Immunological Methods
https://www.readbyqxmd.com/read/28433580/description-of-a-novel-multiplex-avidity-assay-for-evaluating-hpv-antibodies
#4
Allison M Brady, Elizabeth R Unger, Gitika Panicker
Limited data exists regarding antibody avidity for human papillomavirus (HPV). We describe development of a multiplex electrochemiluminescent avidity ELISA for four HPV types (HPV 6, 11, 16, 18) by adding a dissociating step to our established multiplex HPV VLP ELISA. Initial experiments exploring ammonium thiocyanate, sodium thiocyanate and guanidine hydrochloride (GuHCl) as dissociating agents identified GuHCl as most promising. Dissociation conditions with GuHCl were varied (concentration, incubation time, temperature) to select conditions with minimal impact on VLP integrity as measured with monoclonal antibodies to conformational epitopes...
April 19, 2017: Journal of Immunological Methods
https://www.readbyqxmd.com/read/28433579/distinguishing-septic-from-normal-donors-by-detection-of-spla2-iia-from-human-plasma-using-a-microsieve-based-immunoassay
#5
Daniel R Zweitzig, Arjan G Tibbe, Mark J Kopnitsky, Kathleen Cichonski, Leon W M M Terstappen
Bloodstream infections that progress to septic shock are responsible for hundreds of thousands of deaths each year, and are associated with significant healthcare costs. Recent studies have shown that a member of the secreted phospholipase protein family, termed sPLA2-IIA, may play a role during the innate immune response to bacterial infections, and is elevated in the plasma of septic patients. In this report, the feasibility of a simple microsieve-based sPLA2-IIA detection immunoassay was explored. Microsieves containing 5μm pores were covalently coupled with a sPLA2-IIA-specific monoclonal antibody at 0...
April 19, 2017: Journal of Immunological Methods
https://www.readbyqxmd.com/read/28414024/novel-high-throughput-cell-based-hybridoma-screening-methodology-using-the-celigo-image-cytometer
#6
Haohai Zhang, Leo Li-Ying Chan, William Rice, Nasim Kassam, Maria Serena Longhi, Haitao Zhao, Simon C Robson, Wenda Gao, Yan Wu
Hybridoma screening is a critical step for antibody discovery, which necessitates prompt identification of potential clones from hundreds to thousands of hybridoma cultures against the desired immunogen. Technical issues associated with ELISA- and flow cytometry-based screening limit accuracy and diminish high-throughput capability, increasing time and cost. Conventional ELISA screening with coated antigen is also impractical for difficult-to-express hydrophobic membrane antigens or multi-chain protein complexes...
April 14, 2017: Journal of Immunological Methods
https://www.readbyqxmd.com/read/28392436/an-oral-administration-of-a-recombinant-anti-tnf-fusion-protein-is-biologically-active-in-the-gut-promoting-regulatory-t-cells-results-of-a-phase-i-clinical-trial-using-a-novel-oral-anti-tnf-alpha-based-therapy
#7
Einat Almon, Tawfik Khoury, Ariel Drori, Svetlana Gingis-Velitski, Sari Alon, Raul Chertkoff, Mordechai Mushkat, Yoseph Shaaltiel, Yaron Ilan
BACKGROUND: An orally administered BY-2 plant cell-expressed recombinant anti-TNF fusion protein (PRX-106) consists of the soluble form of the human TNF receptor (TNFR) fused to the Fc component of a human IgG1 domain. Aim This study aim at determining the safety and the immune modulatory effect of an oral administration of PRX-106 in humans. METHODS: Three different doses (2, 8 or 16mg/day) of PRX-106 were orally administered for five consecutive days in 14 healthy volunteered participants...
April 6, 2017: Journal of Immunological Methods
https://www.readbyqxmd.com/read/28390927/a-novel-hemolytic-complement-sufficient-nsg-mouse-model-supports-studies-of-complement-mediated-antitumor-activity-in-vivo
#8
Mohit K Verma, Julia Clemens, Lisa Burzenski, Stephen B Sampson, Michael A Brehm, Dale L Greiner, Leonard D Shultz
Monoclonal antibodies (mAbs) have emerged as a mainstream therapeutic option against cancer. mAbs mediate tumor cell-killing through several mechanisms including complement-dependent cytotoxicity (CDC). However, studies of mAb-mediated CDC against tumor cells remain largely dependent on in vitro systems. Previously developed and widely used NOD-scid IL2rγ(null) (NSG) mice support enhanced engraftment of many primary human tumors. However, NSG mice have a 2-bp deletion in the coding region of the hemolytic complement (Hc) gene, and it is not possible to evaluate CDC activity in NSG mice...
April 6, 2017: Journal of Immunological Methods
https://www.readbyqxmd.com/read/28390926/rhodopsin-in-plasma-from-patients-with-diabetic-retinopathy-development-and-validation-of-digital-elisa-by-single-molecule-array-simoa-technology
#9
Eva Rabing Brix Petersen, Dorte Aalund Olsen, Henry Christensen, Søren Berndt Hansen, Cramer Christensen, Ivan Brandslund
BACKGROUND: Diabetic retinopathy (DR) is the most frequent cause of blindness among younger adults in the western world. No blood biomarkers exist to detect DR. Hypothetically, Rhodopsin concentrations in blood has been suggested as an early marker for retinal damage. The aim of this study was therefore to develop and validate a Rhodopsin assay by employing digital ELISA technology, and to investigate whether Rhodopsin concentrations in diabetes patients with DR are elevated compared with diabetes patients without DR...
April 5, 2017: Journal of Immunological Methods
https://www.readbyqxmd.com/read/28390925/proteomic-analysis-of-human-plasma-and-peripheral-blood-mononuclear-cells-in-systemic-lupus-erythematosus-patients
#10
Sara Pinna, Sara Pasella, Marta Deiana, Angela Baralla, Andrea Mannu, Antonio Giovanni Emilio Masala, Piera Veronica Pileri, Nicola Deiana, Fabrizio Scognamillo, Carlo Pala, Angelo Zinellu, Ciriaco Carru, Luca Deiana
Systemic Lupus Erythematosus (SLE) is a systemic autoimmune disease with a broad spectrum of clinical presentations and incompletely understood pathogenesis. This autoimmune disease is characterized by alterations in both the innate and adaptive immune system that lead to the loss of immunologic tolerance. In autoimmune diseases particularly in SLE, early diagnosis, flare or remission phases can be difficult to identify. Proteomics can help to find new therapeutic targets and it also could help to better understand the cellular mechanisms...
April 5, 2017: Journal of Immunological Methods
https://www.readbyqxmd.com/read/28389175/validation-of-a-multi-analyte-panel-with-cell-bound-complement-activation-products-for-systemic-lupus-erythematosus
#11
Thierry Dervieux, John Conklin, Jo-Anne Ligayon, Leilani Wolover, Tyler O'Malley, Roberta Vezza Alexander, Arthur Weinstein, Claudia A Ibarra
BACKGROUND: We describe the analytical validation of an assay panel intended to assist clinicians with the diagnosis of systemic lupus erythematosus (SLE). The multi-analyte panel includes quantitative assessment of complement activation and measurement of autoantibodies. METHODS: The levels of the complement split product C4d bound to erythrocytes (EC4d) and B-lymphocytes (BC4d) (expressed as mean fluorescence intensity [MFI]) are measured by quantitative flow cytometry, while autoantibodies (inclusive of antinuclear and anti-double stranded DNA antibodies) are determined by immunoassays...
April 5, 2017: Journal of Immunological Methods
https://www.readbyqxmd.com/read/28389174/a-biotin-drug-extraction-and-acid-dissociation-bead-procedure-to-eliminate-matrix-and-drug-interference-in-a-protein-complex-anti-drug-antibody-ada-isotype-specific-assay
#12
Hongmei Niu, Thomas Klem, Jinsong Yang, Yongchang Qiu, Luying Pan
Monitoring anti-drug antibody (ADA) responses in patients receiving protein therapeutics treatment is an important safety assessment for regulatory agencies, drug manufacturers, clinicians and patients. Recombinant human IGF-1/IGFBP-3 (rhIGF-1/rhIGFBP-3) is a 1:1 formulation of naturally occurring protein complex. The individual IGF-1 and IGFBP-3 proteins have multiple binding partners in serum matrix with high binding affinity to each other, which presents challenges in ADA assay development. We have developed a biotin-drug extraction with acid dissociation (BEAD) procedure followed by an electrochemiluminescence (ECL) direct assay to overcome matrix and drug interference...
April 4, 2017: Journal of Immunological Methods
https://www.readbyqxmd.com/read/28373028/a-rapid-simple-and-accurate-plaque-assay-for-human-respiratory-syncytial-virus-hrsv
#13
Kyung Sook Kim, Ah Ra Kim, Ying Piao, Ju-Hie Lee, Fu-Shi Quan
Plaque assays of human respiratory syncytial virus (HRSV) are time-consuming, requiring 4 to 7 days for plaque formation and several hours for dye staining. Here, we describe a simple method by which RSV plaques can be visualized and counted with the naked eye only 2 days after infection of HEp-2 cells. In this assay, the infected cells are stained with monoclonal antibodies and the plaques are developed using diaminobenzidine (DAB). We tested the accuracy of this new plaque assay by comparing the results obtained on days 1, 2, 3, 4, 5, and 6 post-infection...
March 31, 2017: Journal of Immunological Methods
https://www.readbyqxmd.com/read/28366645/t-cell-activation-and-proliferation-following-acute-exercise-in-human-subjects-is-altered-by-storage-conditions-and-mitogen-selection
#14
Jacob A Siedlik, Jake A Deckert, Stephen H Benedict, Anuja Bhatta, Amanda J Dunbar, John P Vardiman, Philip M Gallagher
Recent work investigating exercise induced changes in immunocompetence suggests that some of the ambiguity in the literature is resultant from different cell isolation protocols and mitogen selection. To understand this effect, we compared post-exercise measures of T cell activation and proliferation using two different stimulation methods (costimulation through CD28 or stimulation with phytohaemagglutinin [PHA]). Further, we investigated whether exercise induced changes are maintained when T cell isolation from whole blood is delayed overnight in either a room temperature or chilled (4°C) environment...
March 30, 2017: Journal of Immunological Methods
https://www.readbyqxmd.com/read/28365328/comparison-of-6b11-mab-and-%C3%AE-galcer-loaded-cd1d-dextramers-for-detection-of-inkt-cells-by-flow-cytometry
#15
Marzena Lenart, Anna Gruca, Anna Mueck, Magdalena Rutkowska-Zapała, Marta Surman, Anna Szaflarska, Krzysztof Kobylarz, Jarosław Baran, Maciej Siedlar
Invariant natural killer T (iNKT) cells are a small population of thymus-derived T cells that are restricted by non-classical MHC class I molecule CD1d and express an evolutionary conserved TCR with an invariant α-chain. The frequency of iNKT cells in peripheral blood is very low, thus, accurate methods to identify and enumerate iNKT cells are needed. The aim of the study was to compare 6B11 mAb or α-GalCer-loaded CD1d dextramers usage in iNKT cell detection. The frequency of CD3(+)CD56(+) lymphocytes is much higher, with statistical significance (p<0,001), than real iNKT cells detected by 6B11 mAb or α-GalCer-loaded CD1d dextramers...
March 30, 2017: Journal of Immunological Methods
https://www.readbyqxmd.com/read/28365329/lot-to-lot-stability-of-antibody-reagents-for-flow-cytometry
#16
Sebastian Böttcher, Vincent H J van der Velden, Neus Villamor, Matthias Ritgen, Juan Flores-Montero, Hugo Murua Escobar, Tomas Kalina, Monika Brüggemann, Georgiana Grigore, Marta Martin-Ayuso, Quentin Lecrevisse, Carlos E Pedreira, Jacques J M van Dongen, Alberto Orfao
The fluorescence detected using fluorochrome-labelled monoclonal antibodies depends not only on the abundance of the target antigen, but amongst many other factors also on the effective fluorochrome-to-antibody ratio. The diagnostic approach of the EuroFlow consortium relies on reproducible fluorescence intensities over time. A capture bead system for mouse immunoglobulin light chains was utilized to compare the mean fluorescence intensity of 1323 consecutive antibody lots to the currently used lot of the same monoclonal antibody...
March 29, 2017: Journal of Immunological Methods
https://www.readbyqxmd.com/read/28365327/polypropylene-microtitre-plates-modified-with-cr-oh-6-3-for-enhanced-elisa-sensitivity
#17
Nicholas G Welch, Cedric J Lebot, Christopher D Easton, Judith A Scoble, Paul J Pigram, Benjamin W Muir
Chromium solutions have been used as wet chemical modifiers for polymer microtitre plates used in improving immunoassay performance. However, polypropylene has been excluded from the list of potentially modifiable substrates (AnteoTechnologies, 2015). Here we show that untreated polypropylene microtitre plates can indeed be modified using a [Cr(OH)6](3-) complex. Compared to unmodified polypropylene, the chromium modified surfaces demonstrate an up to 4-fold improvement in both assay sensitivity and signal intensity in an antigen capture ELISA...
March 29, 2017: Journal of Immunological Methods
https://www.readbyqxmd.com/read/28341440/optimization-and-testing-of-dried-antibody-tube-the-euroflow-lst-and-pidot-tubes-as-examples
#18
V H J van der Velden, J Flores-Montero, Martin Perez, Marta Martin-Ayuso, Oliver Crespo, Elena Blanco, Tomas Kalina, Jan Philippé, Carolien Bonroy, Maaike de Bie, Jeroen Te Marvelde, Cristina Teodosio, Alba Corral Mateos, Veronika Kanderová, Mirjam van der Burg, Dennis Van Hoof, Jacques J M van Dongen, Alberto Orfao
Within EuroFlow, we recently developed screening tubes for hematological malignancies and immune deficiencies. Pipetting of antibodies for such 8-color 12-marker tubes however is time-consuming and prone to operational mistakes. We therefore evaluated dried formats of the lymphocytosis screening tube (LST) and of the primary immune deficiency orientation tube (PIDOT). Both tubes were evaluated on normal and/or on patient samples, comparing the mean fluorescence intensity of specific lymphocyte populations. Our data show that the dried tubes and liquid counterparts give highly comparable staining results, particularly when analyzed in multidimensional plots...
March 21, 2017: Journal of Immunological Methods
https://www.readbyqxmd.com/read/28336395/human-immune-compartment-comparisons-optimization-of-proliferative-assays-for-blood-and-gut-t-lymphocytes
#19
Jeffrey Dock, Lance Hultin, Patricia Hultin, Julie Elliot, Otto O Yang, Peter A Anton, Beth D Jamieson, Rita B Effros
The accumulation of peripheral blood late-differentiated memory CD8 T cells with features of replicative (cellular) senescence, including inability to proliferate in vitro, has been extensively studied. Importantly, the abundance of these cells is directly correlated with increased morbidity and mortality in older persons. Of note, peripheral blood contains only 2% of the total body lymphocyte population. By contrast, the gut-associated lymphoid tissue (GALT) is the most extensive lymphoid organ, housing up to 60% of total body lymphocytes, but has never been assessed with respect to senescence profiles...
March 21, 2017: Journal of Immunological Methods
https://www.readbyqxmd.com/read/28336396/tumor-control-index-as-a-new-tool-to-assess-tumor-growth-in-experimental-animals
#20
William L Corwin, Hakimeh Ebrahimi-Nik, Stephanie M Floyd, Pouya Tavousi, Ion I Mandoiu, Pramod K Srivastava
Measurement of tumor diameters, tumor volumes, or area under the curve has been traditionally used to quantitate and compare tumor growth curves in immune competent as well as immune-compromised mice and rats. Here, using tumor growth data from a large number of mice challenged with live tumor cells, we describe the use of a new composite parameter, Tumor Control Index (TCI) as an alternative method to do the same. This index, comprised of three distinct values, the Tumor Inhibition Score, Tumor Rejection Score, and Tumor Stability Score, provides a complete picture of nearly every aspect of tumor growth in large numbers of animals, can be deduced automatically from tumor diameter or volume data, and can be used to compare several groups of animals in different experiments...
March 20, 2017: Journal of Immunological Methods
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