ELISA Polyreactivity Antibody Screening

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^{26 (link)}. To detect the polyreactivity of antibodies, the ELISA plate was coated with ssDNA, dsDNA and LPS and Insulin with the final concentration of 10 μg/ml for ssDNA, dsDNA, and LPS, and 5 μg/ml for Insulin in PBS, 50 μl/well, overnight at RT. After blocking for 2 h at RT, properly diluted HmAb64 (50 μl/well) were incubated for 2 h at RT. HRP-conjugated goat anti-human IgG (Jackson, 109-035-098) in dilution buffer (1:1000, 50 μl/well) was incubated for 1 h at RT. At last, the plate was developed for 5 min at 37 °C in 100 µl of a 3,3′5,5′-tetramethylbenzidine substrate solution (Sigma). The reaction was stopped with 50 µl of 2 N H₂SO₄. Plates were read at 450 nm.