Standardization of a cytometric p24-capture bead-assay for the detection of main HIV-1 subtypes

•Identified a cross-reactive antibody to p24 of HIV-1 main subtypes and 2CRFs.•Developed a p24 Luminex capture assay and tested against 56HIV-1 isolates.•Compared the performance of the assay with other quantitative methods.•The assay is highly sensitive with the limit of detection at 3.7pg/ml.•Successfully tested to detect virus production in tissue culture supernatant. The prevailing method to assess HIV-1 replication and infectivity is to measure the production of p24 Gag protein by enzyme-linked immunosorbent assay (ELISA). Since fluorescent bead-based technologies offer a broader dynamic range and higher sensitivity, this study describes a p24 capture Luminex assay capable of detecting HIV-1 subtypes A–D, circulating recombinant forms (CRF) CRF01_AE and CRF02_AG, which together are responsible for over 90% of HIV-1 infections worldwide. The success of the assay lies in the identification and selection of a cross-reactive capture antibody (clone 183-H12-5C). Fifty-six isolates that belonged to six HIV-1 subtypes and CRFs were successfully detected with p-values below 0.021; limits of detection ranging from 3.7 to 3×104pg/ml. The intra- and inter-assay variation gave coefficient of variations below 6 and 14%, respectively. The 183-bead Luminex assay also displayed higher sensitivity of 91% and 98% compared to commercial p24 ELISA and a previously described Luminex assay. The p24 concentrations measured by the 183-bead Luminex assay showed a significant correlation (R=0.92, p