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Journal of Immunological Methods

Fangming Xiu, Zubair Sabz Ali, Nades Palaniyar, Neil Sweezey
New procedures are required to optimize the use of blood samples to study different cell types. The purification of neutrophils and T cells from the same blood sample is not commonly described. We have previously used PolymorphPrep™ (P) or LymphoPrep™ (L) for purifying neutrophils or T cells, respectively. In this study, we describe a new method for purifying both of these cells using P and L from the same sample, and methodological considerations required to obtain consistent Th17 differentiation results...
February 13, 2019: Journal of Immunological Methods
Fleur S van de Bovenkamp, Ninotska I L Derksen, Pleuni Ooijevaar-de Heer, Theo Rispens
About 15% of immunoglobulin G (IgG) molecules contain glycans linked to the antigen-binding fragments (Fab arms) in addition to the glycans linked to the crystallizable fragment (Fc tail) of all IgGs. Fab glycosylation appears to be an important feature of antibodies, for example by influencing antigen binding and antibody stability. The reliable generation of antibodies that either have or lack Fab glycans would be very helpful to study the role of Fab glycans in more detail. In this study, we set out to remove Fab glycans by treating polyclonal and monoclonal human IgG antibodies with two commonly used glycosidases and an improved version of one of the two (Endo F3, PNGase F, and Rapid™ PNGase F)...
February 8, 2019: Journal of Immunological Methods
Rooban B Nahomi, Sandip K Nandi, Ram H Nagaraj
αB-Crystallin is a member of the small heat shock protein family. It is a molecular chaperone and an anti-apoptotic protein. Previous studies have shown that the peptide (73 DRFSVNLDVKHFSPEELKVKV93 , hereafter referred to as peptain-1) from the core domain of αB-crystallin exhibits both chaperone and anti-apoptotic properties similar to the parent protein. We developed a mouse monoclonal antibody against peptain-1 with the aim of blocking the functions of αB-crystallin. The antibody reacted with peptain-1, it did not react with the chaperone peptide of αA-crystallin...
February 6, 2019: Journal of Immunological Methods
Miho Sekai, Jianwei Wang, Nagahiro Minato, Yoko Hamazaki
A clonogenic assay system for thymic epithelial cells (TECs) is of crucial importance for identifying thymic epithelial stem and/or progenitor cells, evaluating their activities, and understanding the mechanisms of thymic involution. However, current systems are not sufficiently sensitive at detecting and quantifying TEC colonies from the adult thymus. Here, we optimized the culture condition to detect visible colonies from adult TECs by modifying our previous culture methods. Epidermal growth factor and leukemia inhibitory factor significantly enhanced the colony-forming efficiency of total TECs from embryo as well as adult mice when added 3 days after plating...
February 6, 2019: Journal of Immunological Methods
Dana Dohr, Robby Engelmann, Brigitte Müller-Hilke
OBJECTIVE: The central mechanism for establishing a self-tolerant and functional T cell repertoire includes the promiscuous expression of otherwise tissue-restricted proteins by medullary thymic epithelial cells (TEC). We here demonstrate a novel and highly efficient method for isolating this rare key cell type. METHODS: We combined the enrichment of medullary TEC via UEA-1 MicroBeads with the subsequent depletion of residual CD45+ hematopoietic cells via specific size exclusion and compared our results to the standard Percoll enrichment method and isolation procedure via flow cytometric cell sorting...
February 5, 2019: Journal of Immunological Methods
Julia A Guzova, Michael J Primiano, Aiping Jiao, Jeffrey Stock, Chiachin Lee, Aaron R Winkler, J Perry Hall
The NLRP3 inflammasome is a complex multimeric signaling apparatus that regulates production of the pro-inflammatory cytokine IL-1β. To overcome both the variability among primary immune cells and the limitations of genetic manipulation of differentiated human or murine macrophages, we developed a simplified, reliable and relevant cell-based model for studying the NLRP3 inflammasome using the undifferentiated human myelomonocytic cell line THP1. Undifferentiated THP1 cells constitutively express NLRP3, and NLRP3 inflammasome activation occurred in response to canonical NLRP3 activation stimuli including nigericin, ATP, and urea crystals, culminating in pro-IL-1β cleavage, extracellular release of mature IL-1β, and pyroptosis...
February 5, 2019: Journal of Immunological Methods
Rachel Tanner, Steven G Smith, Krista E van Meijgaarden, Federico Giannoni, Morven Wilkie, Lucia Gabriele, Carla Palma, Hazel M Dockrell, Tom H M Ottenhoff, Helen McShane
A major challenge to TB vaccine development is the lack of a validated immune correlate of protection. Mycobacterial growth inhibition assays (MGIAs) represent an unbiased measure of the ability to control mycobacterial growth in vitro. A successful MGIA could be applied to preclinical and clinical post-vaccination samples to aid in the selection of novel vaccine candidates at an early stage and provide a relevant measure of immunogenicity and protection. However, assay harmonisation is critical to ensure that comparable information can be extracted from different vaccine studies...
January 30, 2019: Journal of Immunological Methods
L M Kranz, B Gärtner, A Michel, M Pawlita, T Waterboer, N Brenner
By inducing immunosuppression in infected patients, human immunodeficiency virus 1 (HIV-1) generates a favorable environment for opportunistic infections and the development of several human cancers. In order to detect individual serum or plasma HIV-1 antibody status for epidemiological studies, high-throughput HIV-1 Multiplex Serology was developed. Seven HIV-1 antigens were recombinantly expressed in E. coli as N-terminal glutathione-S-transferase (GST) fusion proteins that are bound to glutathione-coupled sets of beads with distinct fluorescent color...
January 17, 2019: Journal of Immunological Methods
Sowmya Nagaraj, Prakash Narayana Reddy, Shylaja Ramlal, Soumya Paul, Bhavani Peddayelachagiri, D M Parida
Antibody cross-reactivity is a problem often associated with closely related antigens. This study was aimed to develop a method enabling differentiation of closely related toxins based on antigen designing strategy. The method involves identification of disparate amino acids (AA) confined to target antigen in comparison with two or more closely related antigens, their assembly into a DNA oligomer and further cloning as six tandem repeats (TR) using restriction and ligation strategy into a desired vector and finally generation of antigen specific antibodies...
January 17, 2019: Journal of Immunological Methods
Anwar M Hashem, Sawsan S Al-Amri, Tagreed L Al-Subhi, Loai A Siddiq, Ahmed M Hassan, Maha M Alawi, Rowa Y Alhabbab, Salwa I Hindawi, Osama B Mohammed, Nabil S Amor, Abdulaziz N Alagaili, Ahmed A Mirza, Esam I Azhar
Since 2012, MERS-CoV has caused up to 2220 cases and 790 deaths in 27 countries with Saudi Arabia being the most affected country with ~83.1% of the cases and ~38.8% local death rate. Current serological assays such as microneutralization (MN), plaque reduction neutralization, immunofluorescence, protein microarray or pseudoparticle neutralization assays rely on handling of live MERS-CoV in high containment laboratories or need for expensive and special equipment and reagents and highly trained personnel which represent a technical hurdle for most laboratories in resource-limited MERS-CoV endemic countries...
January 16, 2019: Journal of Immunological Methods
Christian P Moritz, Yannick Tholance, François Lassablière, Jean-Philippe Camdessanché, Jean-Christophe Antoine
Indirect enzyme-linked immunosorbent assay (ELISA) is an important diagnostic method as it enables the quantification of the presence of autoantibodies in human blood sera. However, unspecific binding of antibodies to the solid phase causes considerable serum-specific background noise (SSBN), involving the risk of false positive diagnosis. Therefore, we present a simple and concise, yet obvious proof-of-principle of a recently suggested normalization method. The method is based on subtracting SSBN by using non-coated ELISA wells as a control for each serum-of-interest...
January 14, 2019: Journal of Immunological Methods
Mohammed S O Tagod, Satoshi Mizuta, Yuki Sakai, Masashi Iwasaki, Kengo Shiraishi, Hiroaki Senju, Hiroshi Mukae, Craig T Morita, Yoshimasa Tanaka
The adoptive transfer of immune effector cells, such as CD8+ killer αβ T cells, γδ T cells, NK (natural killer) cells, and genetically-modified T cells, has been receiving increasing attention. It is essential to determine cellular cytotoxicity so as to monitor the function and quality of ex vivo-expanded immune effector cells before infusion. The most common method is the [51 Cr]-sodium chromate release assay. It is, however, preferable to avoid the use of radioactive materials in clinical laboratories...
January 14, 2019: Journal of Immunological Methods
Se Pyeong Im, Jaesung Kim, Jung Seok Lee, Si Won Kim, Jae Wook Jung, Jassy Mary S Lazarte, Jin Hong Chun, Young Rim Kim, Jong Pyo Suh, Kim D Thompson, Tae Sung Jung
The variable lymphocyte receptor B (VLRB) of jawless vertebrates has a similar function to the antibodies produced by jawed vertebrates, and has been considered as an alternative source to mammalian antibodies for use in biological research. We developed a modified yeast display vector system (pYD8) to display recombinant hagfish VLRB proteins on the extracellular surface of yeast for the isolation of antigen-specific VLRBs. After observing an up-regulation in the VLRB response in hagfish immunized with hemagglutinin 1 of avian influenza virus H9N2 subtype (H9N2-HA1), the antigen-specific VLRBs decorated on the yeast's surface were selected by quantitative library screening through magnetic-activated cell sorting (MACS) and fluorescent-activated cell sorting (FACS)...
January 3, 2019: Journal of Immunological Methods
Josh Poorbaugh, Tanushree Samanta, Stuart W Bright, Sean E Sissons, Ching-Yun Chang, Pankaj Oberoi, Angus J MacDonald, Andrea P Martin, Karen L Cox, Robert J Benschop
IL-21 is a pleiotropic cytokine that plays a key role in modulating inflammatory responses, including the promotion of autoimmune diseases. Several groups have quantitated circulating levels of IL-21 in plasma and serum samples using various commercial ELISAs. We determined, however, that the most commonly used commercial assays in published literature were not specific or sensitive enough to detect levels of IL-21 in heparin plasma or serum from healthy human individuals. This finding prompted an effort to develop more specific and sensitive methods to quantitate IL-21 in complex biological matrices using proprietary anti-IL-21 antibodies with the Quanterix SiMoA platform and the Meso Scale Discovery (MSD) S-PLEX® format...
December 24, 2018: Journal of Immunological Methods
Lauren E Higdon, Corey J Cain, Melissa A Colden, Jonathan S Maltzman
Single cell sequencing has recently been applied to many immunological studies. Flow cytometric index sorting isolates cells for single cell sequencing with protein level data linked to sequences. However, successful sequencing of index sorted samples requires careful optimization of several sort parameters, including nozzle size, flow rate, threshold rate, and yield calculations. In this study, considerations and optimization data for each of these variables are presented. Our analysis focused on index sorting, but the findings can be applied to any plate sorting protocol...
December 24, 2018: Journal of Immunological Methods
Laura M De Plano, Enza Fazio, Maria G Rizzo, Domenico Franco, Santina Carnazza, Sebastiano Trusso, Fortunato Neri, Salvatore P P Guglielmino
Sepsis is a systemic inflammatory response ensuing from presence and persistence of microorganisms in the bloodstream. The possibility to identify them at low concentrations may improve the problem of human health and therapeutic outcomes. So, sensitive and rapid diagnostic systems are essential to evaluate bacterial infections during the time, also reducing the cost. In this study, from random M13 phage display libraries, we selected phage clones that specifically bind surface of Staphyloccocus aureus, Pseudomonas aeruginosa and Escherichia coli...
December 12, 2018: Journal of Immunological Methods
Tomoko Wada, Kiyoshi Hirahara, Ami Aoki, Yuki Morimoto, Masahiro Kiuchi, Jin Kumagai, Mikiko Okano, Kota Kokubo, Miki Kato, Chiharu Fukano, Katsuyo Ohashi-Doi, Toshinori Nakayama
House dust mites (HDMs), Dermatophagoides sp., are one of the most widespread aeroallergens worldwide and cause various allergic diseases, including asthma. The pathophysiology of asthma has been intensively investigated using murine models of allergic airway inflammation induced by exposure to D. pteronyssinus. However, the pathogenic roles of D. farinae in the allergic airway inflammation remains unclear. We herein report that repetitive exposure to D. farinae resulted in neutrophil-dominant airway inflammation together with fibrotic changes and the formation of lymphoid clusters...
December 11, 2018: Journal of Immunological Methods
Axel Petzold
The best validated immunoassays for neurodegeneration have been developed for class III and IV intermediate filaments. There are a number of unique biochemical features of the intrinsically unstructured polyampholytic tail regions of these proteins which affect domain structure and thereby affinity and epitope recognition of antibodies used in immunoassays. Here one of these intermediate filaments, the neurofilament heavy chain, is chosen to demonstrate the effect of the ionic strength of a buffer system on the analytical signal to noise ratio...
December 10, 2018: Journal of Immunological Methods
Soi Cheng Law, Oscar L Haigh, Carina M Walpole, Colm Keane, John Miles, Maher K Gandhi, Kristen J Radford, Raymond J Steptoe
Current HLA-typing methods are typically designed to provide exquisitely-detailed identification of multiple HLA-alleles to satisfy the requirements for organ and bone marrow transplantation or genetic studies. Many human immunological studies, on the other hand, focus around only a small number of HLA alleles that are abundant or of relevance to specific diseases. Consequently, for such studies, many HLA typing approaches are not cost-effective and are potentially complicated, slow and not easily performed in-house...
December 8, 2018: Journal of Immunological Methods
Teppo Salminen, Etvi Juntunen, Sheikh M Talha, Kim Pettersson
Lateral flow (LF) immunoassays are commonly used for point-of-care testing and typically incorporate visually read reporters, such as gold particles. To improve sensitivity and develop quantitative LF immunoassays, visual reporters can be replaced by fluorescent reporters detected by an instrument. In this study, we used fluorescent europium(III) chelate doped nanoparticle (Eu-np) reporters to develop a quantitative high-sensitivity LF immunoassay for free prostate specific antigen (fPSA). Furthermore, we tested different simplified formats of the assay and the effect of different modifiable parameters on the detection limit of the assay: dynamic range, assay duration and number of assay steps...
December 4, 2018: Journal of Immunological Methods
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