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

Abstract

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 × 104 pg/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<0.0001) with the data obtained from quantitative real time PCR. This newly developed p24 assay leverages the advantages of the Luminex platform, which include smaller sample volume and simultaneous detection of up to 500 analytes in a single sample, and delivers a valuable tool for the field.

Keywords: Detection; HIV-1; Multi-subtype; Multiplex assay; p24.

Figure 1

Validation of the coupling reaction by flow cytometry. The different coupled microspheres (183-beads, empty circles; 39/5.4A-beads, empty squares; IG-beads, empty diamonds and 4F6-beads, empty triangles) were mixed with serial dilutions of PE-labeled detection antibody (6.25-200μg/ml) and assessed by flow cytometry. The graph represents the fluorescence intensity depending on the amount of detection antibody.

Figure 2

Validation of the functionality of the capture antibodies post coupling. The capture microspheres (183-beads, empty circles; 39/5.4A-beads, empty squares; IG-beads, empty diamonds and 4F6-beads, empty triangles) were mixed with serial dilutions of purified p24 protein, the binding was revealed by PE-labeled anti-p24 and read by Luminex. The graph represents standard curves fitted by a logistic 5P regression using Prism6.

Figure 3

p24 standard curve using the clone 183-H12-5C. The graph represents the mean fluorescence intensity of a standard curve run in quadruplicate fitted by a logistic 5P regression using Prism6, with R²=0.9993. The CV of the data is indicated below each data point..

Figure 4

p24 capture Luminex assay of the 56 isolates representing 6 HIV-1 subtypes. The graph represents the mean concentration (in pg/ml) of each sample ran in quadruplicate. The error bars represent the SEM (Standard Error of the Mean).

Figure 5

Inter-assay variation. p24 capture Luminex assay of the 30 isolates representing 6 HIV-1 subtypes. The graph represents the mean concentrations (in pg/ml) of 30 isolates run in duplicates in four idependant experiments carried out over a period of time of several months. The error bars represent the SEM.

Figure 6

Comparison of p24 concentration of the 56 HIV-1 isolates between the capture Luminex assay and the commercial ELISA kit. The concentrations are expressed in pg/ml. The empty circles represent the isolates for which the commercial ELISA kit measured higher p24 concentration than the Luminex capture assay.

Figure 7

p24 capture Luminex assay using anti-p24gag HIV-1 mAb clone 4F6 (full circles), clone 183-H12-5C (empty circles)and ELISA kit (empty squares). The graph represents the p24 titration (in pg/ml) of 12 HIV-1 virus isolates representing six different subtypes (A, B, C, D, CRF01_AE, CRF01_AG).