Optimization and validation of a neutralizing antibody assay for HIV-1 in A3R5 cells
2014
Sarzotti-Kelsoe, Marcella | Daniell, Xiaoju | Todd, Christopher A. | Bilska, Miroslawa | Martelli, Amanda | LaBranche, Celia | Perez, Lautaro G. | Ochsenbauer, Christina | Kappes, John C. | Rountree, Wes | Denny, Thomas N. | Montefiori, David C.
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.
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