Optimization and validation of a neutralizing antibody assay for HIV-1 in A3R5 cells.
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A3R5 is a human CD4(+) lymphoblastoid cell line that was engineered to express CCR5 and is useful for the detection of weak neutralizing antibody responses against tier 2 strains of HIV-1. Here we describe the optimization and validation of the HIV-1 neutralizing antibody assay that utilizes A3R5 cells, performed in compliance with Good Clinical Laboratory Practice (GCLP) guidelines. The assay utilizes Renilla luciferase-expressing replication competent infectious molecular clones (IMC) encoding heterologous env genes from different HIV-1 clades. Key assay validation parameters tested included specificity, accuracy, precision, limit of detection and quantitation, specificity, linearity and range, and robustness. Plasma samples demonstrated higher non-specific activity than serum samples in the A3R5 assay. This assay can tolerate a wide range of virus input but is more sensitive to cell concentration. The higher sensitivity of the A3R5 assay in neutralization responses to tier 2 strains of HIV-1 makes it complementary to, but not a substitute for the TZM-bl assay. The validated A3R5 assay is employed as an endpoint immunogenicity test for vaccine-elicited neutralizing antibodies against tier 2 strains of HIV-1, and to identify correlates of protection in HIV-1 vaccine trials conducted globally.
High-Throughput Screening Assays
Limit of Detection
Practice Guidelines as Topic
Predictive Value of Tests
Reproducibility of Results
Published Version (Please cite this version)10.1016/j.jim.2014.02.013
Publication InfoSarzotti-Kelsoe, M; Daniell, X; Todd, CA; Bilska, M; Martelli, A; LaBranche, C; ... Montefiori, DC (2014). Optimization and validation of a neutralizing antibody assay for HIV-1 in A3R5 cells. J Immunol Methods, 409. pp. 147-160. 10.1016/j.jim.2014.02.013. Retrieved from https://hdl.handle.net/10161/14688.
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Thomas Norton Denny
Professor in Medicine
Thomas N. Denny, MSc, M.Phil, is the Chief Operating Officer of the Duke Human Vaccine Institute (DHVI), Associate Dean for Duke Research and Discovery @RTP, and a Professor of Medicine in the Department of Medicine at Duke University Medical Center. He is also an Affiliate Member of the Duke Global Health Institute. Previously, he served on the Health Sector Advisory Council of the Duke University Fuquay School of Business. Prior to joining Duke, he was an Associate Professor of Pathology, Labo
Celia Crane LaBranche
Associate Professor Emeritus
David Charles Montefiori
Professor in Surgery
Dr. Montefiori is Professor and Director of the Laboratory for HIV and COVID-19 Vaccine Research & Development in the Department of Surgery, Division of Surgical Sciences at Duke University Medical Center. His major research interests are viral immunology and HIV and COVID-19 vaccine development, with a special emphasis on neutralizing antibodies. Multiple aspects of HIV-1 neutralizing antibodies are studied in his laboratory, including mechanisms of neutralization and escape,
Research Professor of Integrative Immunobiology
Ongoing Applied Activities •I direct a Global Quality Assurance Program, which I developed and pioneered here at Duke University, to oversee compliance with Good Clinical Laboratory Practice Guidelines in three HIV vaccine trial networks (CHAVI, CAVD, Duke HVTN, EQAPOL, Duke VTEU) involving domestic and international laboratory sites. •I also direct a Global Proficiency Testing Program for laboratories testing for neutralizing antibody function in individuals infected
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