Optimization and validation of a neutralizing antibody assay for HIV-1 in A3R5 cells.
Abstract
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.
Type
Journal articleSubject
A3R5 cellsAssay validation
HIV
Neutralizing antibody
Antibodies, Neutralizing
Automation, Laboratory
Biomarkers
Cell Line
Guideline Adherence
HIV Antibodies
HIV Infections
HIV-1
High-Throughput Screening Assays
Humans
Limit of Detection
Neutralization Tests
Observer Variation
Practice Guidelines as Topic
Predictive Value of Tests
Quality Control
Reproducibility of Results
Time Factors
Transfection
Permalink
https://hdl.handle.net/10161/14688Published Version (Please cite this version)
10.1016/j.jim.2014.02.013Publication Info
Sarzotti-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.This is constructed from limited available data and may be imprecise. To cite this
article, please review & use the official citation provided by the journal.
Collections
More Info
Show full item recordScholars@Duke
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,
Marcella Sarzotti-Kelsoe
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
Alphabetical list of authors with Scholars@Duke profiles.

Articles written by Duke faculty are made available through the campus open access policy. For more information see: Duke Open Access Policy
Rights for Collection: Scholarly Articles
Works are deposited here by their authors, and represent their research and opinions, not that of Duke University. Some materials and descriptions may include offensive content. More info