Relative epitope mapping of monoclonal antibodies against HIV gag p24 for sandwich ELISA of HIV-1 detection

Abstract

Monoclonal antibodies (MAb) against HIV-1 Gag p24 were produced by hybridomas fused with spleen cells of mice immunized with purified Gag and SP2/O-Agl4 myeloma. Fifteen hybridomas producing anti-Gag MAb were isolated. Of them, seven MAb reacting with p24 in Western blot analysis were used for epitope mapping of Gag. Four MAb (4C9, 4E5, 5E4 and 2A11) of them were biotinylated and incubated with diluted MAb in microtiter plate wells coated with Gag. The results of competitive inhibition were detected by avidin-horse radish peroxidase ELISA system. Our results showed that 4C9, 4C8 and 4E5 recognize the same epitope, while 5E4 and 1B2 recognize distinctly different epitopes. Sandwich one-step ELISA for detecting HIV l Gag p24, a major core antigen, was estabilished utilizing 1B2 as capture antibody and 4C9 as biotinylated antibody. Sensitivity of our assay system was 150 pg/ml when HIV-1 lysate was used as an antigen, while it showed 40 pg/ml with HIV-1 Gag p24 as an antigen.