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Journal of Immunological Methods

Kazuhiro Taguchi, Takashi Onoe, Tomoaki Yoshida, Yoshinori Yamashita, Kiyomi Taniyama, Hideki Ohdan
Tumor endothelial cells (TECs), which constitute the lining of the tumor blood vessels, have various characteristics as tumor constituent cells. In this study, we describe a novel method for the isolation of highly pure, fresh TECs, which form a small population within the tumor. Tumors were first dissected from tumor-bearing mice and digested to a single cell suspension with Collagenase Type II; the single cells were then separated by density gradient centrifugation. TECs were enriched by CD31-positive selection using magnetic activated cell sorting and subsequently purified by fluorescence activated cell sorting...
November 2, 2018: Journal of Immunological Methods
Yang Song, Julie Nigro, Lijia Yu, Mario Congiu, Narelle Skinner, Alexander Thompson, Kumar Visvanathan
Cytokine production by human peripheral blood mononuclear cells including monocytes, is frequently assessed by measuring secreted cytokines using enzyme linked immunosorbent assay (ELISA), whereby the total concentration of one cytokine of interest is obtained without information regarding the cell type responsible for making the cytokine. Cytokines can be retained inside the cell using protein transport inhibitors. Subsequent analysis by flow cytometry not only identifies the cell type producing the cytokine but can semi-quantitate the amount of cytokine produced by measuring the geometric mean fluorescence intensity (gMFI) and is amenable to analyzing more than one protein associated with the same cell (multiplexing)...
November 2, 2018: Journal of Immunological Methods
Liisa Murray, Yang Xi, John W Upham
Plasmacytoid dendritic cells (pDC) are an important type I interferon producer that play an important role in the first line of host defence during viral infection. Abnormalities in pDC numbers and function have been associated with several health conditions. Quantifying pDC is important for understanding pDC related immune responses in viral infections and other diseases, however the current methods for quantifying pDC using flow cytometry have limited utility in large cohort studies involving multiple centres with limited access to flow cytometry...
November 2, 2018: Journal of Immunological Methods
Edith González-González, Rosa Camacho-Sandoval, Alexis Jiménez-Uribe, Alejandra Montes-Luna, Ilselena Cortés-Paniagua, Jazmín Sánchez-Morales, Leslie Muñoz-García, Alejandra V Tenorio-Calvo, Carlos A López-Morales, Marco A Velasco-Velázquez, Lenin Pavón, Sonia Mayra Pérez-Tapia, Emilio Medina-Rivero
The development of biotherapeutics requires continuous improvement in analytical methodologies for the assessment of their quality attributes. A subset of biotherapeutics is designed to interact with specific antigens that are exposed on the membranes of target cells or circulating in a soluble form, and effector functions are achieved via recognition of their Fc region by effector cells that induce mechanisms such as antibody-dependent cell-mediated cytotoxicity (ADCC). Thus, ADCC induction is a critical quality attribute (CQA) that must be evaluated to ensure biotherapeutic efficacy...
November 2, 2018: Journal of Immunological Methods
Haoran Gao, Yining Jin, Dan Loan Jian, Eric Olson, Perry K W Ng, Venu Gangur
BACKGROUND: Wheat allergy is a major food allergy that has reached significant levels of global public health concern. Potential variation in allergenicity among different wheat genotypes is not well studied at present largely due to the unavailability of validated methods. Here, we developed and validated a novel mouse-based primary screening method for this purpose. METHODS: Groups of Balb/c mice weaned on-to a plant protein-free diet were sensitized with salt-soluble protein (SSP) extracted from AABB genotype of wheat (durum, Carpio variety)...
November 2, 2018: Journal of Immunological Methods
Miho Tanaka, Akiko Ishige, Masami Yaguchi, Takehisa Matsumoto, Mikako Shirouzu, Shigeyuki Yokoyama, Issay Kitabayashi, Toshitada Takemori, Michishige Harada
Antibody-based therapeutic strategies have become recognized as useful clinical options in several types of cancer, often with the expectation that such therapies will trigger target cell elimination via antibody-dependent cellar cytotoxicity (ADCC) by natural killer cells. The successful development of therapeutic monoclonal antibodies (mAbs) requires an assay system that permits a critical evaluation of their physicochemical and biological characteristics. At present a number of ADCC assay systems have been reported, however, there is still room for improvement in terms of usability, operability and sensitivity...
October 30, 2018: Journal of Immunological Methods
Nami Iwamoto, Rosemarie Mason, Jianfei Hu, Amy Ransier, Hugh Welles, Kaimei Song, Daniel Douek, Mario Roederer
An effective prophylactic vaccine against human immunodeficiency virus (HIV) will likely require a potent antibody response that can neutralize the virus at the mucosal portal of entry. The elicitation of potent broadly-neutralizing anti-sera will be an iterative process, optimizing candidates that only block a fraction of potential viral strains. This effect, termed "sieving", is evidence of a partially efficacious vaccine. Understanding the mechanisms of resistance of the breakthrough viruses is important for improving vaccines...
October 30, 2018: Journal of Immunological Methods
Fiyaz Mohammed, Daniel H Stones, Benjamin E Willcox
X-ray crystallographic studies of class I peptide-MHC molecules (pMHC) continue to provide important insights into immune recognition, however their success depends on generation of diffraction-quality crystals, which remains a significant challenge. While protein engineering techniques such as surface-entropy reduction and lysine methylation have proven utility in facilitating and/or improving protein crystallisation, they risk affecting the conformation and biochemistry of the class I MHC antigen binding groove...
October 24, 2018: Journal of Immunological Methods
Amanda L Wooster, Trevor S Anderson, Devin B Lowe
MHC class I-specific reagents such as fluorescently-labeled multimers (e.g., tetramers) have greatly advanced the understanding of CD8+ T cells under normal and diseased states. However, recombinant MHC class I components (comprising MHC class I heavy chain and β2 microglobulin) are usually produced in bacteria following a lengthy purification protocol that requires additional non-covalent folding steps with exogenous peptide for complete molecular assembly. We have provided an alternative and rapid approach to generating soluble and fully-folded MHC class I molecules in eukaryotic cell lines (such as CHO cells) using a Sleeping Beauty transposon system...
October 19, 2018: Journal of Immunological Methods
Benjamin D Groff, Andrew W L Kinman, Jacob F Woodroof, Rebecca R Pompano
Explants of lymphoid tissue provide a rare opportunity to assess the organization of the immune system in a living, dynamic environment. Traditionally, ex vivo immunostaining is conducted in fixed tissue sections, while live tissues are analyzed using genetically engineered fluorescent reporters or adoptively transferred, pre-labelled cell populations. Here, we validated a protocol for immunostaining and imaging in live, thick slices of lymph node tissue, thus providing a spatial "map" of the lymph node while maintaining the viability and functionality of the slices...
October 19, 2018: Journal of Immunological Methods
Najib Aziz, Anthony W Butch, Timothy C Ryner, Otoniel Martinez-Maza, Roger Detels
BACKGROUND: The use of anticoagulants may influence the composition of blood cells and interfere with plasma levels of IL-1ra when unprocessed EDTA blood samples are stored for long periods of time. METHODS: Blood was drawn into EDTA and heparinized blood collection tubes from 11 HIV-1 negative men participating in the Multicenter AIDS Cohort Study (MACS) and 4 healthy volunteers. The blood was processed according to the experiments detailed in the method and after incubation; supernatants were collected and stored at -70 °C until batch testing using IL-1ra ELISA...
October 19, 2018: Journal of Immunological Methods
Marie Senant, Pauline Bordereau, Eric Tartour, Marie-Agnès Dragon-Durey
The detection and the quantification of specific antibodies represent essential tools for the diagnosis and for the biological monitoring of immune humoral response in many clinical situations in particular in autoimmune diseases or in the context of immunotherapy using monoclonal antibodies. This article focuses on the development of a specific antibody measuring method (Patent n°PCT/IB2014/064437). The principle of this method is based on the combined use of a monoclonal antibody as standard and the protein G as immunoglobulins detecting agent...
October 18, 2018: Journal of Immunological Methods
Lina Chakrabarti, Arshia Mathew, Lina Li, Soojin Han, Judith Klover, Tom Albanetti, Pamela Hawley-Nelson
Development of cell lines for biotherapeutic protein production requires screening large numbers of clones to identify and isolate high producing ones. As such, stable cell line generation is a time- and resource-intensive process. There is an increasing need to enhance the selection efficiency of high-yielding clonal cell lines for cell line development projects by using high throughput screening of live cells for markers predictive of productivity. Single cell deposition by fluorescence activated cell sorting (FACS) is a commonly performed method for cloning to generate cell lines derived from a single recombinant cell...
October 17, 2018: Journal of Immunological Methods
Mari Kono, Katsuyasu Saigo, Shiori Matsuhiroya, Takayuki Takahashi, Makoto Hashimoto, Ayako Obuchi, Shion Imoto, Takashi Nishiyama, Seiji Kawano
Neutrophils are recruited to infection sites and kill bacteria by phagocytosis and reactive oxygen species (ROS) production. It has been reported that vacuoles are present in neutrophils that produce ROS and are present in large numbers in blood smears of patients with bacterial infections. The leukocyte differentiation function on the Sysmex automated hematology analyzer classifies leukocytes by flow cytometry. Particularly, side-scattered light is known to reflect the quantity of organelles. This study investigated the possibility of detecting vacuoles or invagination of cell membrane in neutrophils producing ROS using a hematology analyzer...
October 16, 2018: Journal of Immunological Methods
Nicholas A Zwang, Balaji B Ganesh, Kim T Cardenas, Anita S Chong, Patricia W Finn, David L Perkins
BACKGROUND AND PURPOSE: Phospho flow cytometry is a powerful technique to analyze signaling in rare cell populations. This technique, however, requires harsh conditions for cell fixation and permeabilization, which can denature surface antigens or antibody-conjugated fluorochromes. These are among several technical limitations which have been a barrier to quantify signaling in unique B cell subsets. One such immature subset, transitional B cells (TrBs), may play a role in suppressing solid organ transplant rejection, graft-versus-host disease, autoimmunity, and even the immune response to malignancy...
October 13, 2018: Journal of Immunological Methods
Caroline Murphy, Edwina Stack, Svetlana Krivelo, Mark Breheny, Hui Ma, Richard O'Kennedy
Antibody-based sensors are now widely used in therapeutics, diagnostics, and in environmental monitoring. Recombinant antibodies are becoming integral parts of such devices due to their reported high affinities, their capacity for engineering to achieve highly defined performance characteristics and the fact that their production can be optimized to a significant degree. To aid as a model for the identification of important analyte binding residues within the antibody sub-structure and elucidate the docking characteristics of small molecules such as metabolites, illicit drugs, biotherapeutics (proteins, peptides and nucleic acids) or toxins towards the antibody, herein we report the binding of the harmful cyanobacterial-toxin, microcystin-leucine-arginine (MC-LR) to a single chain fragment variable (scFv) antibody fragment...
October 12, 2018: Journal of Immunological Methods
Joel S Simon, Sergio Botero, Sanford M Simon
The adaptive immune response generates a large repertoire of T cells with T-cell receptors (TCRalpha and TCRbeta ) and B cells with immunoglobulins (Ig). The repertoire changes in response to antigen stimulation both through amplification of specific cells (clonal expansion) as well as somatic hypermutation of immunoglobulins. Alterations of the immune repertoire have been observed in response to acute disease, such as external pathogens, or chronic diseases, such as autoimmunity and cancer. Here we establish experimental and analytical protocols for quantifying the peripheral blood of healthy human individuals by profiling the immune repertoire for the Complementarity determining region 3 (CDR3) of the variable regions of TCRbeta (CDRβ3) and the IgG heavy chain (CDRH1, CDRH2, CDRH3)...
October 10, 2018: Journal of Immunological Methods
Robert J Kubiak, Jianchun Zhang, Pin Ren, Harry Yang, Lorin K Roskos
Cut point determination is an important aspect of immunogenicity assay development. The cut point can be influenced by a myriad of factors. Key among those is the analytical variability of the assay itself and biological variation due to test samples. Since a smaller cut point value may result in improved sensitivity, the existing procedures often employ statistical techniques such as outlier removal to produce a conservative cut point. Although such practices are intended to yield acceptable assay sensitivity, they may fail to fully account for biological variability in the data, thus generating higher than expected number of false positive results...
October 9, 2018: Journal of Immunological Methods
Stanley J Naides
Accelerated identification of autoantibodies associated with previously idiopathic neurological disease has provided insights into disease mechanisms, enhanced understanding of neurological function, and opportunities for improved therapeutic interventions. The role of the laboratory in the expanding field of neuroimmunology is critical as specific autoantibody identification provides guidance to clinicians in diagnosis, prognosis, tumor search strategies, and therapeutic interventions. The number of specific autoantibodies identified continues to increase and newer testing strategies increase efficiencies in the laboratory and availability to clinicians...
October 6, 2018: Journal of Immunological Methods
Chia Sharpe, Joanne Davis, Kylie Mason, Constantine Tam, David Ritchie, Rachel Koldej
Understanding how cancer and cancer therapies affect the immune system is integral to the rational application of immunotherapies. Flow cytometry is the gold standard method of peripheral blood immune cell profiling. However, the requirement for viable cells can limit its applicability, especially in studies of retrospective clinical cohorts. We aimed to determine if gene expression, analysed using the NanoString platform, could be used to quantify the immune populations present in cryopreserved peripheral blood mononuclear cell (PBMC) samples from patients with chronic lymphocytic leukaemia...
September 26, 2018: Journal of Immunological Methods
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