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Journal of Immunological Methods

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https://www.readbyqxmd.com/read/28238798/unambiguous-high-resolution-genotyping-of-human-leukocyte-antigens
#1
Robert A Bradshaw, Paul P J Dunn
We have developed a high resolution sequencing based typing method for genotyping Human Leukocyte Antigens (HLA) over a period of twenty years. The methods are based upon the separation of HLA alleles per locus at the initial amplification to simplify the analysis post-sequencing. The increasing discovery of polymorphism in HLA, manifested in new alleles, has necessitated the continuing development of this method. Here we present methods for the high resolution Sequence Based Typing of HLA-A, B, C (class I) and HLA-DQB1 and DRB1 (class II)...
February 23, 2017: Journal of Immunological Methods
https://www.readbyqxmd.com/read/28237707/study-of-fluid-dynamics-reveals-direct-communications-between-lymphatic-vessels-and-venous-blood-vessels-at-lymph-nodes-of-mice
#2
Kazu Takeda, Shiro Mori, Tetsuya Kodama
Cancer cells metastasize to lymph nodes, with distant metastasis resulting in poor prognosis. The role of lymph node metastasis (LNM) in the spread of cancer to distant organs remain incompletely characterized. The visualization of flow dynamics in the lymphatic and blood vessels of MXH10/Mo-lpr/lpr mice, which develop systemic swelling of lymph nodes up to 10mm in diameter, has revealed that lymph nodes have the potential to be a direct source of systemic metastasis. However, it is not known whether these fluid dynamics characteristics are universal phenomena present in other strains of laboratory mice...
February 22, 2017: Journal of Immunological Methods
https://www.readbyqxmd.com/read/28216237/development-of-an-anti-ferret-cd4-monoclonal-antibody-for-the-characterisation-of-ferret-t-lymphocytes
#3
Daniel S Layton, Xiaowen Xiao, John D Bentley, Louis Lu, Cameron R Stewart, Andrew G D Bean, Timothy E Adams
The ferret is an established animal model for a number of human respiratory viral infections, such as influenza virus and more recently, Ebola virus. However, a paucity of immunological reagents has hampered the study of cellular immune responses. Here we describe the development and characterisation of a novel monoclonal antibody (mAb) against the ferret CD4 antigen and the characterisation of ferret CD4 T lymphocytes. Recombinant production and purification of the ferret CD4 ectodomain soluble protein allowed hybridoma generation and the generation of a mAb (FeCD4) showing strong binding to ferret CD4 protein and lymphoid cells by flow cytometry...
February 16, 2017: Journal of Immunological Methods
https://www.readbyqxmd.com/read/28216236/elimination-of-falsely-reactive-results-in-a-commercially-available-west-nile-virus-igm-capture-enzyme-linked-immunosorbent-assay-by-heterophilic-antibody-blocking-reagents
#4
Harry E Prince, Mary Lapé-Nixon, Tara S Givens, Tiffany Bradshaw, Marek J Nowicki
All sera initially reactive in the Focus Diagnostics West Nile virus IgM capture enzyme-linked immunosorbent assay (WNV IgM ELISA) must be retested with background subtraction to identify falsely-reactive (FR) samples due to antibodies that bind to immunoglobulins of other animal species (heterophilic antibodies). In some settings, such as pre-transplant testing of organ donors, the reporting delay associated with retesting can have an adverse impact on donor procurement and organ placement. We sought to determine if inclusion of heterophilic antibody blockers in assay conjugate could eliminate the nonspecific reactivity of FR samples...
February 16, 2017: Journal of Immunological Methods
https://www.readbyqxmd.com/read/28212879/flow-cytometric-based-protocols-for-assessing-anti-mt-2-igg1-reactivity-high-dimensional-data-handling-to-define-predictors-for-clinical-follow-up-of-human-t-cell-leukemia-virus-type-1-infection
#5
Jordana Grazziela Coelho-Dos-Reis, Vanessa Peruhype-Magalhães, Marcelo Antônio Pascoal-Xavier, Matheus de Souza Gomes, Laurence Rodrigues do Amaral, Ludmila Melo Cardoso, Juan Jonathan-Gonçalves, Ágata Lopes Ribeiro, Ana Lúcia Borges Starling, João Gabriel Ribas, Denise Utsch Gonçalves, Anna Bárbara de Freitas Carneiro-Proietti, Andréa Teixeira-Carvalho, Olindo Assis Martins-Filho
The present work provides an innovative methodological approach to assess the anti-HTLV-1 IgG1 reactivity with practical application in clinical laboratory. Serum from non-infected healthy controls (NI) and HTLV-1-infected patients, categorized as asymptomatic (AS), putatively progressing to HTLV-1 associated myelopathy/tropical spastic paraparesis - HAM/TSP (pHAM) or with clinical diagnosis of HAM/TSP (HT) were assayed in two-parallel flow cytometry platforms, referred as: Fix and Fix&Perm protocols. Operating-characteristics analysis indicated that a single pair of attributes ("serum dilution/cut-off") for Fix and Fix&Perm protocols presented excellent performance for the diagnosis of HTLV-1 infection...
February 14, 2017: Journal of Immunological Methods
https://www.readbyqxmd.com/read/28212880/automatic-detection-of-hypoxia-in-renal-tissue-stained-with-hif-1alpha
#6
Aline Rodrigues Buzin, Nayana Damiani Macedo, Isabela Bastos Binotti Abreu De Araujo, Breno Valentim Nogueira, Tadeu Uggere de Andrade, Denise Coutinho Endringer, Dominik Lenz
OBJECTIVE: The objective of this study was the identification of the stain HIF-alpha using the Image Cytometry, and to help to count the positive cells (with HIF-alpha) and the negative cells (without HIF-alpha) from the same sample. METHOD: 17 images of renal tissues from male rats of Winstar lineage; overall, there were 12.587 objects (cells) in the images for analysis. The acquired images were then analyzed through the free softwares CellProfiler (version 2.1...
February 14, 2017: Journal of Immunological Methods
https://www.readbyqxmd.com/read/28209381/an-easy-fast-and-low-tech-equipment-requiring-alternative-method-to-optimize-immunolabelling-conditions-for-pre-embedding-immunogold-electron-microscopy-and-to-correlate-light-and-electron-microscopical-immunogold-labelling-results
#7
Shweta Suiwal, Gabriele Kiefer, Frank Schmitz, Karin Schwarz
Correlating light microscopic immunolabelling results with electron microscopic data is of great interest in many fields of biomedical research but typically requires very specialized, expensive equipment and complex procedures which are not available in most labs. In this technical study, we describe an easy and "low-tech"-equipment-requiring pre-embedding immunolabelling approach that allows correlation of light microscopical immunolabelling results with electron microscopic (EM) data as demonstrated by the example of immunolabelled synaptic ribbons from retinal rod photoreceptor synapses...
February 13, 2017: Journal of Immunological Methods
https://www.readbyqxmd.com/read/28189705/magnetic-sorting-of-membrane-associated-igg-for-phenotype-based-selection-of-stable-antibody-producing-cells
#8
Y Poitevin, G Pontini, N Fischer, M Kosco-Vilbois, G Elson
To establish a simple and widely accessible technique for rapidly selecting high producing Chinese hamster ovary (CHO) cells engineered to express a monoclonal antibody (mAb), we have exploited the transient display of recombinant protein on their cell surface. In combination with magnetic bead-based methods, we demonstrate the ability to select for cells of high productivity in the absence of any metabolic-based selection method. This technique is sufficient to obtain genetically stable engineered CHO cells via a single step of cell subcloning and yields sought-after stable, high IgG producing clonal cell lines...
February 8, 2017: Journal of Immunological Methods
https://www.readbyqxmd.com/read/28174050/a-novel-antihuman-c3d-monoclonal-antibody-with-specificity-to-the-c3d-complement-split-product
#9
Karina Juhl Rasmussen, Mikkel-Ole Skjodt, Lars Vitved, Karsten Skjoedt, Yaseelan Palarasah
The complement component C3 and the cleavage products of C3b/iC3b, C3c and C3d are used as biomarkers in clinical diagnostics. Currently, no specific antibodies are able to differentiate C3d from other fragments, although such a distinction could be very valuable considering that they may reflect different pathophysiological mechanisms. We have developed a rat antihuman C3d monoclonal antibody with specificity to the end sequence of the N-terminal region of C3d. The antibody can therefore only bind to C3d when it manifests itself as the final end product of cleaved C3...
February 4, 2017: Journal of Immunological Methods
https://www.readbyqxmd.com/read/28167276/detection-and-monitoring-pla2r-autoantibodies-by-lips-in-membranous-nephropathy
#10
Peter D Burbelo, Laurence H Beck, Meryl Waldman
Autoantibodies against the M-type phospholipase A2 receptor (PLA2R) are specific markers for primary membranous nephropathy (MN). Quantification of PLA2R autoantibodies is an important, noninvasive tool that facilitates the diagnosis and monitoring of primary MN. In this report we describe a highly quantitative luciferase immunoprecipitation systems (LIPS) assay for detecting PLA2R autoantibodies. For these studies, a cDNA fragment encoding the first 858 amino acids of PLA2R protein was cloned to generate N-terminal antigen fusion constructs with Gaussia luciferase (Gluc) and Nano luciferase (NanoLuc) reporters...
February 4, 2017: Journal of Immunological Methods
https://www.readbyqxmd.com/read/28167277/purification-of-recombinant-c-reactive-protein-mutants
#11
Avinash Thirumalai, Sanjay K Singh, David J Hammond, Toh B Gang, Donald N Ngwa, Asmita Pathak, Alok Agrawal
C-reactive protein (CRP) is an evolutionarily conserved protein, a component of the innate immune system, and an acute phase protein in humans. In addition to its raised level in blood in inflammatory states, CRP is also localized at sites of inflammation including atherosclerotic lesions, arthritic joints and amyloid plaque deposits. Results of in vivo experiments in animal models of inflammatory diseases indicate that CRP is an anti-pneumococcal, anti-atherosclerotic, anti-arthritic and an anti-amyloidogenic molecule...
February 3, 2017: Journal of Immunological Methods
https://www.readbyqxmd.com/read/28167275/single-amino-acid-fingerprinting-of-the-human-antibody-repertoire-with-high-density-peptide-arrays
#12
Laura K Weber, Andrea Palermo, Jonas Kügler, Olivier Armant, Awale Isse, Simone Rentschler, Thomas Jaenisch, Jürgen Hubbuch, Stefan Dübel, Alexander Nesterov-Mueller, Frank Breitling, Felix F Loeffler
The antibody species that patrol in a patient's blood are an invaluable part of the immune system. While most of them shield us from life-threatening infections, some of them do harm in autoimmune diseases. If we knew exactly all the antigens that elicited all the antibody species within a group of patients, we could learn which ones correlate with immune protection, are irrelevant, or do harm. Here, we demonstrate an approach to this question: First, we use a plethora of phage-displayed peptides to identify many different serum antibody binding peptides...
February 3, 2017: Journal of Immunological Methods
https://www.readbyqxmd.com/read/28167274/characterization-of-human-leukocyte-huvec-adhesion-effect-of-cell-preparation-methods
#13
Franca Marino, Laura Schembri, Emanuela Rasini, Monica Pinoli, Angela Scanzano, Alessandra Luini, Terenzio Congiu, Marco Cosentino
OBJECTIVE: Sample manipulation to obtain isolated granulocytes represents a key, and often necessary, step in the in vitro studies. We investigated by the means of flow cytometry and microscopic techniques (both optical microscopy [OM] and scanning electron microscopy [SEM]), the granulocyte-endothelium adhesion and the role of sample manipulation. METHODS: By means of a co-culture method, we have analysed the adhesion of human leukocytes, originated from two different blood samples (fresh venous blood [FB] and buffy coat [BC]), to the human umbilical venous endothelial cell (HUVEC) monolayer...
February 3, 2017: Journal of Immunological Methods
https://www.readbyqxmd.com/read/28163018/multiplexed-fc-array-for-evaluation-of-antigen-specific-antibody-effector-profiles
#14
Eric P Brown, Karen G Dowell, Austin W Boesch, Erica Normandin, Alison E Mahan, Thach Chu, Dan H Barouch, Chris Bailey-Kellogg, Galit Alter, Margaret E Ackerman
Antibodies are widely considered to be a frequent primary and often mechanistic correlate of protection of approved vaccines; thus evaluating the antibody response is of critical importance in attempting to understand and predict the efficacy of novel vaccine candidates. Historically, antibody responses have been analyzed by determining the titer of the humoral response using measurements such as an ELISA, neutralization, or agglutination assays. In the simplest case, sufficiently high titers of antibody against vaccine antigen(s) are sufficient to predict protection...
February 2, 2017: Journal of Immunological Methods
https://www.readbyqxmd.com/read/28159551/antibody-mediated-depletion-of-immunosuppressive-factors-from-ovarian-carcinoma-associated-ascites-for-investigation-of-paracrine-versus-autocrine-effects
#15
E Brencicova, S S Diebold
Many studies seek to explore the impact of extrinsic soluble factors present in serum, interstitial fluids or cell-conditioned media on cells in vitro. A convenient approach to elucidate the effects of a particular factor is its selective neutralization. However, intrinsic production of soluble factors such as cytokines by the cultured cells is common and can have an impact via autocrine mechanisms. The addition of cytokine-specific neutralizing antibodies leads to neutralization of the targeted factors irrespective of their source and affects paracrine and autocrine effects alike...
January 31, 2017: Journal of Immunological Methods
https://www.readbyqxmd.com/read/28159550/characterization-of-the-diversity-of-t-cell-receptor-%C3%AE-%C3%AE-complementary-determinant-region-3-in-human-peripheral-blood-by-immune-repertoire-sequencing
#16
Hui Chen, Mingjin Zou, Da Teng, Jianmin Zhang, Wei He
γδ T cells function as sentinels in early host response to infections and malignancies. Although γδ T cells are regarded as innate immune cells and recognize antigens in a non-MHC restricted manner, they possess a huge diversity of complementary determinant region 3 (CDR3) of T cell receptor (TCR) generated by the rearrangement of germ-line gene V- (D) -J-C fragments. However, the detailed characteristics of the TCRγδ CDR3 repertoire remain unclear. A comprehensive analysis would answer fundamental questions about the diversity of the TCRγδ CDR3 repertoire and elucidate the mechanism underlying γδ T cell recognition of pathogens and tumor antigens...
January 31, 2017: Journal of Immunological Methods
https://www.readbyqxmd.com/read/28131818/improved-production-of-single-domain-antibodies-with-two-disulfide-bonds-by-co-expression-of-chaperone-proteins-in-the-escherichia-coliperiplasm
#17
Lisa C Shriver-Lake, Ellen R Goldman, Daniel Zabetakis, George P Anderson
Single domain antibodies are recombinantly expressed variable domains derived from camelid heavy chain antibodies. Natural single domain antibodies can have noncanonical disulfide bonds between their complementarity-determining regions that help position the binding site. In addition, engineering a second disulfide bond serves to tie together β-sheets thereby inhibiting unfolding. Unfortunately, the additional disulfide bond often significantly decreases yield, presumably due to formation of incorrect disulfide bonds during the folding process...
January 25, 2017: Journal of Immunological Methods
https://www.readbyqxmd.com/read/28130033/efficient-evaluation-of-humoral-immune-responses-by-the-use-of-serum-pools
#18
Louise Sternbæk, Anette H Draborg, Christoffer T Nielsen, Søren Jacobsen, Line V Iversen, Lone Troelsen, Elke Theander, Gunnar Houen
BACKGROUND: Collection and testing of individual serum samples are often used in research to gain knowledge about e.g. the humoral response against bacteria or virus. This is a valid but time-consuming method and might be a waste of valuable serum samples for inefficient research. So far, no study has considered using serum pools as a quick and efficient screening method to confirm or deny hypotheses. METHODS: We created serum pools from four different patient groups (systemic lupus erythematosus n=85, rheumatoid arthritis n=77, Sjögren's syndrome n=91, systemic sclerosis n=66) and one healthy control group (n=67)...
January 25, 2017: Journal of Immunological Methods
https://www.readbyqxmd.com/read/28109682/label-free-checkerboard-assay-to-determine-overlapping-epitopes-of-ebola-virus-vp-40-antibodies-using-surface-plasmon-resonance
#19
George P Anderson, Jinny L Liu, Dan Zabetakis, Patricia M Legler, Ellen R Goldman
Immunoassay formats, in which antibodies provide sensitivity and specificity, are often utilized to provide rapid and simple diagnostic tests. Surface plasmon resonance is frequently used to evaluate the suitability of antibodies by determining binding kinetics to agents or surrogate antigens. We used SPR to evaluate a number of commercial monoclonal antibodies as well as single domain antibodies produced in-house. All the antibodies targeted the Ebola virus viral protein 40 (VP40). We determined the ability of each antibody to bind to immobilized VP40, and ensured they did not bind Ebola glycoprotein or the nucleoprotein...
January 19, 2017: Journal of Immunological Methods
https://www.readbyqxmd.com/read/28093271/characterization-and-clinical-validation-of-mcm2-and-top2a-monoclonal-antibodies-in-the-bd-proex%C3%A2-c-assay-an-immunoassay-which-detects-aberrant-s-phase-induction-in-cervical-tissue
#20
Eric P Dixon, Lorraine M King, Ramona Nelson, Stephen G Simkins, Steven L Knapp, George H Brough, Karen L Lenz, Dorian T Henderson, Clark M Whitehead, Janice Hessling, Charlotte A Brown, Douglas P Malinowski
BACKGROUND: The Papanicolaou (Pap) screen has been successful in reducing cervical cancer; but exhibits low sensitivity when detecting cervical dysplasia. Use of molecular biomarkers in Pap tests may improve diagnostic accuracy. DESIGN: Monoclonal antibodies to Minichromosome Maintenance Protein 2 (MCM2) and DNA Topoisomerase II α (TOP2A) were selected for use in IHC based on their ability to differentiate normal from diseased cervical tissues in tissue microarrays...
January 16, 2017: Journal of Immunological Methods
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