Binding Specificity of HIV-1 Antibody

ELISA was conducted to examine the HIV-1 Env-specific bindings in DP6-001 serum samples, EBV-transformed B cell media and the final mAb64 against gp120, gp120-Core or their D368R mutant protein as previously described ^{22 (link)}. To detect the polyreactivity of monoclonal antibodies, ELISA plate was coated with ssDNA, dsDNA and LPS and Insulin with final concentration of 10μg/ml for ssDNA, dsDNA and LPS, and 5μg/ml for Insulin in PBS, 50 μl/well, overnight at RT. After blocking for 2 hours at RT, properly diluted HmAb64 (50μl/well) were Incubated for 2 hours at RT. HRP-conjugated goat anti-human IgG (Jackson, 109-035-098) in dilution buffer (1:1000, 50 μl/well) was incubated for 1 hour at RT. At last, plate was developed for 5 minutes at 37°C in 100 µl of a 3,3’5,5’-tetramethylbenzidine substrate solution (Sigma). The reaction was stopped with 50 µl of 2N H2SO4. Plates were read at 450 nm.