Figure 1
Quantitative p17 secretion assay.
(A) Sensitivity of p17 antigen capture ELISA. To detect and quantify recombinant p17, the viral protein was resuspended in complete medium at different concentrations then added to anti-p17 mAb MBS15-coated ELISA microwells. Immunocomplexes were revealed using biotinylated mAb MBS34 followed by peroxidase-labelled streptavidin. Assay specificity was confirmed by the absence of p24 capture by mAb MBS15 when biotinylated anti-p24 mAb MBS-12 was used as a specific reagent in the revealing step. (B) Quantification of p17 released by Jurkat-HIV-1. Evaluation of p17 release in the cell culture supernatant was performed by cellular ELISA. Jurkat cells were nucleofected with or without the pNL4-3 expression vector. After 24 h, Jurkat-HIV-1 were seeded onto ELISA plates pre-coated with anti-p17 mAb MBS15 and incubated further for 16 h to allow protein secretion and accumulation. Quantification of released p17 was performed using a standard curve generated with recombinant p17 as in (A). Amount of secreted p17 was calculated as mean ± SD of eight independent experiments performed in triplicate. The horizontal line in the middle of each box indicates the mean amount of secreted p17, whereas the top and bottom borders of the box mark the 75th and 25th percentiles, respectively (left panel). The release of p17 by Jurkat-Gag cells takes place in the absence of cell disruption as shown by the absence of CFSE in the cellular supernatant of Jurkat-Gag and Jurkat-EGFP. CFSE was quantified by fluorometric technique and 100% of RLU detection was calculated by performing a detergent-based cell disruption (right panel). Significance was assessed using Student’s t test; ***P < 0.001. (C) Detection of Pr55Gag and p17 in cell extracts and cell culture supernatants. Jurkat-Gag cell extracts (left panel) and culture supernatants (right panel) were immunoprecipitated by a rabbit polyclonal antibody to p17 and then analyzed for p17 and Pr55Gag expression by Western blot, using the anti-p17 mAb MBS-3 and the anti-p24 mAb MBS12 as specific reagents. Full-length blots were cropped for final display.
