Optimization and validation of a neutralizing antibody assay for HIV-1 in A3R5 cells.

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2014-07

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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.

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10.1016/j.jim.2014.02.013

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Sarzotti-Kelsoe, M, X Daniell, CA Todd, M Bilska, A Martelli, C LaBranche, LG Perez, C Ochsenbauer, et al. (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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