Sarzotti-Kelsoe, MarcellaDaniell, XiaojuTodd, Christopher ABilska, MiroslawaMartelli, AmandaLaBranche, CeliaPerez, Lautaro GOchsenbauer, ChristinaKappes, John CRountree, WesDenny, Thomas NMontefiori, David C2021-01-042021-01-042014-070022-17591872-7905https://hdl.handle.net/10161/22003A3R5 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.Cell LineHumansHIV-1HIV InfectionsHIV AntibodiesObserver VariationNeutralization TestsReproducibility of ResultsPredictive Value of TestsTransfectionTime FactorsQuality ControlGuideline AdherencePractice Guidelines as TopicAntibodies, NeutralizingHigh-Throughput Screening AssaysAutomation, LaboratoryLimit of DetectionBiomarkersJournal article2021-01-04