1. Optimization and validation of a neutralizing antibody assay for HIV-1 in A3R5 cells.
- Author
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Sarzotti-Kelsoe M, Daniell X, Todd CA, Bilska M, Martelli A, LaBranche C, Perez LG, Ochsenbauer C, Kappes JC, Rountree W, Denny TN, and Montefiori DC
- Subjects
- Automation, Laboratory standards, Biomarkers blood, Cell Line, Guideline Adherence standards, HIV Infections blood, HIV Infections immunology, HIV-1 genetics, Humans, Limit of Detection, Observer Variation, Practice Guidelines as Topic standards, Predictive Value of Tests, Quality Control, Reproducibility of Results, Time Factors, Transfection, Antibodies, Neutralizing blood, HIV Antibodies blood, HIV Infections diagnosis, HIV-1 immunology, High-Throughput Screening Assays standards, Neutralization Tests standards
- 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., (Copyright © 2014 Elsevier B.V. All rights reserved.)
- Published
- 2014
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