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Journal of Immunological Methods

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https://www.readbyqxmd.com/read/28109682/label-free-checkerboard-assay-to-determine-overlapping-epitopes-of-ebola-virus-vp-40-antibodies-using-surface-plasmon-resonance
#1
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 18, 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
#2
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
https://www.readbyqxmd.com/read/28093272/a-method-for-obtaining-simian-immunodeficiency-virus-rna-sequences-from-laser-capture-microdissected-and-immune-captured-cd68-and-cd163-macrophages-from-frozen-tissue-sections-of-bone-marrow-and-brain
#3
Jaclyn Mallard, Emily Papazian, Caroline Soulas, David J Nolan, Marco Salemi, Kenneth C Williams
Laser capture microdissection (LCM) is used to extract cells or tissue regions for analysis of RNA, DNA or protein. Several methods of LCM are established for different applications, but a protocol for consistently obtaining lentiviral RNA from LCM captured immune cell populations is not described. Obtaining optimal viral RNA for analysis of viral genes from immune-captured cells using immunohistochemistry (IHC) and LCM is challenging. IHC protocols have long antibody incubation times that increase risk of RNA degradation...
January 13, 2017: Journal of Immunological Methods
https://www.readbyqxmd.com/read/28063769/immunogenicity-assay-cut-point-determination-using-nonparametric-tolerance-limit
#4
Jianchun Zhang, Wenjia Li, Lorin K Roskos, Harry Yang
The newly released FDA guidance on immunogenicity assay development and validation recommends use of a lower confidence limit of the percentile of the negative subject population as the cut point in order to guarantee a pre-specified false positive rate with high confidence. The limit is, in essence, a lower tolerance limit. Although in literature several methods are available for determining the tolerance limit, they either fail to take into account the repeated measurement of the data from a typical immunogenicity assay quantification/validation experiment or rely heavily on normality assumption of the data, which is rarely correct...
January 4, 2017: Journal of Immunological Methods
https://www.readbyqxmd.com/read/28041941/a-simple-flow-cytometry-method-to-evaluate-peripheral-blood-contamination-of-bone-marrow-aspirates
#5
José Antonio Delgado, Francisco Guillén-Grima, Cristina Moreno, Carlos Panizo, Carmen Pérez-Robles, Juan José Mata, Laura Moreno, Paula Arana, Silvia Chocarro, Juana Merino
Bone marrow (BM) aspirates used for flow-cytometry (FCM) studies are usually obtained from a second aspiration, as the primary aspirate is used for morphological assessment. For this reason, the FCM samples unavoidably contain some blood; although, good-quality samples contain only a small amount. It is of utmost importance to assess the quality of samples prior to FCM analysis; yet, contamination with peripheral blood (PB) is not evaluated in most laboratories, possibly because the methods available are either qualitative or too complex for daily practice...
December 29, 2016: Journal of Immunological Methods
https://www.readbyqxmd.com/read/28039100/utility-of-systematic-isolation-of-immune-cell-subsets-from-hiv-infected-individuals-for-mirna-profiling
#6
Manel E Bargalló, Alberto C Guardo, Maria J Maleno, Laia Miralles, Lander Egaña-Gorroño, Tuixent Escribà, Felipe García, Jose M Gatell, Mireia Arnedo, Montserrat Plana
INTRODUCTION: Peripheral blood mononuclear cells (PBMCs) are frequently used for genomic analyses, but several factors can affect the yield and integrity of nucleic acids, including the methods of cell collection and isolation. The goal of this work was to analyze the utility of systematic isolation of different immune cell subsets by immunomagnetic separation and the RNA integrity after isolated cells from samples of HIV-infected patients. METHODS: PBMC from Healthy Controls (HC, n=15), Elite Controllers (EC, n=15), Viremic Controllers (VC, n=15), Viremic Progressors (VP, n=15) and HIV-infected patients on therapy (ART, n=15) were isolated by Ficoll-Paque density gradient centrifugation...
December 27, 2016: Journal of Immunological Methods
https://www.readbyqxmd.com/read/28034712/evaluation-of-a-digital-dispenser-for-direct-curve-dilutions-in-a-vaccine-potency-assay
#7
Christopher Roselle, Dana Whitehouse, Thy Follmer, Fran Ansbro, Silikhone Bouaraphan, Liming Guan, Sha-Ke Wang, Mary Shank-Retzlaff, Thorsten Verch
Dilutions are a common source of analytical error, both in terms of accuracy and precision, and a common source of analyst mistakes. When serial dilutions are used, errors compound, even when employing laboratory automation. Direct point dilutions instead of serial dilutions can reduce error but is often impractical as they require either large diluent volumes or very small sample volumes when performed with traditional liquid handling equipment. We evaluated preparation of dilution curves using a picoliter digital dispenser, the HP, Inc...
December 27, 2016: Journal of Immunological Methods
https://www.readbyqxmd.com/read/28024998/development-and-validation-of-a-cell-based-fluorescent-method-for-measuring-antibody-affinity
#8
Xin Yu, Charles N Pegram, Darell D Bigner, Vidyalakshmi Chandramohan
Monoclonal antibodies have become essential tools for diagnostic and therapeutic purposes. Antibody affinity is one of the critical factors influencing the therapeutic success of tumor-targeting antibodies. Therefore, developing an accurate and reliable method for determining antibody affinity is crucial. In this study, we describe a fluorescent cell-based immunosorbent assay that can accurately measure antibody affinity (KD) in the nanomolar range. This method involves the addition of fluorescently labeled antibodies to antigen-positive and antigen-negative cell lines fixed on 96-well plates...
December 24, 2016: Journal of Immunological Methods
https://www.readbyqxmd.com/read/27939301/accurate-quantitation-for-in-vitro-refolding-of-single-domain-antibody-fragments-expressed-as-inclusion-bodies-by-referring-the-concomitant-expression-of-a-soluble-form-in-the-periplasms-of-escherichia-coli
#9
Tomoaki Noguchi, Yuichi Nishida, Keiji Takizawa, Yue Cui, Koki Tsutsumi, Takashi Hamada, Yoshisuke Nishi
Single domain antibody fragments from two species, a camel VHH (PM1) and a shark VNAR (A6), were derived from inclusion bodies of E. coli and refolded in vitro following three refolding recipes for comparing refolding efficiencies: three-step cold dialysis refolding (TCDR), one-step hot dialysis refolding (OHDR), and one-step cold dialysis refolding (OCDR), as these fragments were expressed as 'a soluble form' either in cytoplasm or periplasm, but the amount were much less than those expressed as 'an insoluble form (inclusion body)' in cytoplasm and periplasm...
December 8, 2016: Journal of Immunological Methods
https://www.readbyqxmd.com/read/27939300/characterization-of-fc%C3%AE-riiia-effector-cells-used-in-in-vitro-adcc-bioassay-comparison-of-primary-nk-cells-with-engineered-nk-92-and-jurkat-t-cells
#10
Yao-Te Hsieh, Poonam Aggarwal, David Cirelli, Ling Gu, Teresa Surowy, Ned M Mozier
Antibody-dependent cell-mediated cytotoxicity (ADCC) is an important mechanism of action (MOA) of several therapeutic antibody drugs and evaluation in ADCC bioassays is important in antibody drug development and maintenance. Three types of effector cells now routinely used in bioassay evaluation of ADCC are natural killer cells from human donors (FcγRIIIA+primary NK), FcγRIIIA engineered NK-92 cells and FcγRIIIA/NFAT-RE/luc2 engineered Jurkat T cells. Engineered effector cells were developed to address need for improved precision and accuracy of classic NK cell ADCC bioassays...
December 8, 2016: Journal of Immunological Methods
https://www.readbyqxmd.com/read/27923642/development-of-ifn-%C3%AE-secretory-elispot-based-assay-for-screening-of-adcc-responses
#11
Ashwini Shete, Poonam Suryawanshi, Chetan Chavan, Archana Kulkarni, Sheela Godbole, Manisha Ghate, Madhuri Thakar
Antibody dependent cell mediated cytotoxicity has been established as one of the important protective immune mechanisms against HIV making it essential to evaluate it while testing immunogenicity of emerging vaccine candidates. IFN-γ secretory ELISPOT assay, widely used for evaluation of CTL response in HIV vaccine trials, was adapted for measuring ADCC responses and the results were compared with the standard ICS based assays. IFN-γ responses elicited by plasma samples of 23 HIV infected individuals against Env and Gag peptides using granulocytes as antigen presenting cells were assessed by both the methods...
December 5, 2016: Journal of Immunological Methods
https://www.readbyqxmd.com/read/27919837/ex-vivo-itreg-differentiation-revisited-convenient-alternatives-to-existing-strategies
#12
Billur Akkaya, Amanda H Holstein, Christopher Isaac, Mitra P Maz, Deborah D Glass, Ethan M Shevach, Munir Akkaya
Ex-vivo differentiation of regulatory T cells (Tregs) from naïve CD4(+) T-cells has been widely used in immunological research. Isolation of a highly pure naïve T cell population is the key factor that determines the efficiency of subsequent Treg differentiation. Currently, this step relies mostly on FACS sorting, which is often costly, time consuming, and inconvenient. Alternatively, magnetic separation of T-cells can be performed; yet, available protocols fail to reach sort level purity and consequently result in low Treg differentiation efficiency...
December 2, 2016: Journal of Immunological Methods
https://www.readbyqxmd.com/read/27913143/innate-immune-stimulatory-activity-of-porphyromonas-gingivalis-fimbriae-is-eliminated-by-phase-separation-using-triton-x-114
#13
Kohji Nozoe, Terukazu Sanui, Masaaki Takeshita, Takao Fukuda, Akira Haraguchi, Yoshitomi Aida, Fusanori Nishimura
Fimbriae are virulence factors of Porphyromonas gingivalis (P. gingivalis). In this study, the action of fimbriae on neutrophil respiratory burst and cytokine production by mononuclear cells (MNC) were investigated. Native or denatured form of purified P. gingivalis fimbriae contained endotoxin at an equivalence of 1-3μglipopolysaccharides(LPS)/mg protein. The endotoxin could be reduced to the equivalent of 1ng-LPS/mg protein by phase separation using Triton X-114. Unfractionated fimbriae caused serum-dependent priming of neutrophils for enhanced respiratory burst, but both native and denatured forms of Triton X-114-fractionated fimbriae were not active at 100μg/mL...
November 30, 2016: Journal of Immunological Methods
https://www.readbyqxmd.com/read/27889562/systematic-evaluation-of-monoclonal-antibodies-and-immunoassays-for-the-detection-of-interferon-%C3%AE-and-interleukin-2-in-old-and-new-world-non-human-primates
#14
Ankie Höglind, Irene Areström, Cecilia Ehrnfelt, Khosro Masjedi, Bartek Zuber, Luis Giavedoni, Niklas Ahlborg
Non-human primates (NHP) provide important animal models for studies on immune responses to infections and vaccines. When assessing cellular immunity in NHP, cytokines are almost exclusively analyzed utilizing cross-reactive anti-human antibodies. The functionality of antibodies has to be empirically established for each assay/application as well as NHP species. A rational approach was employed to identify monoclonal antibodies (mAb) cross-reactive with many NHP species. Panels of new and established mAbs against human Interferon (IFN)-γ and Interleukin (IL)-2 were assessed for reactivity with eukaryotically expressed recombinant IFN-γ and IL-2, respectively, from Old (rhesus, cynomolgus and pigtail macaques, African green monkey, sooty mangabey and baboon) and New World NHP (Ma's night monkey, squirrel monkey and common marmoset)...
November 24, 2016: Journal of Immunological Methods
https://www.readbyqxmd.com/read/27889561/inhibition-of-interleukin-5-induced-false-positive-anti-drug-antibody-responses-against-mepolizumab-through-the-use-of-a-competitive-blocking-antibody
#15
Karen Liao, Erik Meyer, Thomas N Lee, Amy Loercher, Daniel Sikkema
Mepolizumab, a humanized IgG1 monoclonal antibody that blocks native homodimeric interleukin-5 (IL-5) from binding to the IL-5 receptor, has recently been approved for treatment of severe eosinophilic asthma. Our initial immunogenicity assay method for phase I and II studies utilized a bridging electrochemiluminescence format with biotin and ruthenium-labelled mepolizumab linked by anti-drug antibodies (ADA). We discovered that IL-5 significantly increased in dosed subjects from a phase II study and that the increased IL-5 was in the form of a drug-bound complex...
November 24, 2016: Journal of Immunological Methods
https://www.readbyqxmd.com/read/27856192/nk-cell-degranulation-as-a-marker-for-measuring-antibody-dependent-cytotoxicity-in-neutralizing-and-non-neutralizing-human-sera-from-dengue-patients
#16
Peifang Sun, Brian J Morrison, Charmagne G Beckett, Zhaodong Liang, Nishith Nagabhushana, An Li, Kevin R Porter, Maya Williams
The study assessed antibody-dependent NK cell degranulation, a biomarker relevant to antibody-dependent cell cytotoxicity (ADCC), to analyze dengue immune sera. We first determined binding intensity of patient sera to the surface of DENV-infected cells and examined the types of antigens expressed on infected cells. Antigens from pre-membrane (PreM) and envelope (E), but not from NS proteins were detected on the surface of infected cells. After adding NK cells to infected target cells previously treated with patient sera, rapid NK cell degranulation was observed...
November 14, 2016: Journal of Immunological Methods
https://www.readbyqxmd.com/read/27851889/a-novel-method-for-assaying-human-regulatory-t-cell-direct-suppression-of-b-cell-effector-function
#17
Elizabeth Weingartner, Jean-Paul Courneya, Achsah Keegan, Amit Golding
We have established a highly reproducible and reliable protocol for testing human regulatory T cell function in suppressing IgM production from an immature human B cell line. The autoreactive Ramos B cell line provides a stable reporter of B cell effector function that can be tested by a straight-forward IgM ELISA. Tregs from healthy volunteers display a range of ability for suppressing baseline IgM production in a contact- and death-independent manner. Having established the normal range for human Treg direct suppression of B cell effector function, it will now be possible to efficiently test Tregs from various autoimmune conditions in which B cell hyperactivity and secretion of auto-antibodies are a hallmark of disease...
November 13, 2016: Journal of Immunological Methods
https://www.readbyqxmd.com/read/27983956/increasing-the-accuracy-and-scalability-of-the-immunofluorescence-assay-for-epstein-barr-virus-by-inferring-continuous-titers-from-a-single-sample-dilution
#18
Sherry Meow Peng Goh, Muthukaruppan Swaminathan, Julian U-Ming Lai, Azlinda Anwar, Soh Ha Chan, Ian Cheong
High Epstein Barr Virus (EBV) titers detected by the indirect Immunofluorescence Assay (IFA) are a reliable predictor of Nasopharyngeal Carcinoma (NPC). Despite being the gold standard for serological detection of NPC, the IFA is limited by scaling bottlenecks. Specifically, 5 serial dilutions of each patient sample must be prepared and visually matched by an evaluator to one of 5 discrete titers. Here, we describe a simple method for inferring continuous EBV titers from IFA images acquired from NPC-positive patient sera using only a single sample dilution...
January 2017: Journal of Immunological Methods
https://www.readbyqxmd.com/read/27876507/exercise-induced-cytokine-changes-in-antigen-stimulated-whole-blood-cultures-compared-to-serum
#19
C Pilat, K Krüger, T Frech, F C Mooren
Strenuous exercise is followed by an elevation of many cytokines with inflammation regulating properties. Since most cytokines act at pico- or nanomolar concentrations many investigations failed to detect their concentrations in vivo. Hence, the aim of this study was to evaluate the significance of cytokine measurements (IL-1β, TNF-α, IL-1ra, IL-6, CCL2 and CXCL8) in a stimulated whole-blood culture (sWBC) compared to serum with respect to their exercise-induced kinetics and detection rates. 40 male volunteers (age: 25,5±4,3years, BMI: 24,00±2,24, VO2peak: 46,9±4,1mL/kg×min) performed 60min of intensive bicycle exercise (80% VO2peak)...
January 2017: Journal of Immunological Methods
https://www.readbyqxmd.com/read/27876506/fungal-peptides-from-pneumonitis-hypersensitivity-etiologic-agents-are-able-to-induce-specific-cellular-immune-response
#20
Anne-Pauline Bellanger, Thibaud Lignon, Yann Godet, Bénédicte Rognon, Gabriel Reboux, Houssein Gbaguidi-Haore, Christophe Borg, Laurence Millon
PURPOSE: Hypersensitivity pneumonitis (HP) is an immunoallergic disease due to chronic exposure to high quantities of different microorganisms such as Mycobacterium immunogenum (Mi), a mycobacterium, and Lichtheimia corymbifera (Lc), a filamentous fungus. It has recently been demonstrated that the protein DLDH (dihydrolipoyl dehydrogenase), is common to these microorganisms. This study aimed to investigate the immune potential of overlapping peptide pools covering the MiDLDH and LcDLDH...
January 2017: Journal of Immunological Methods
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