Antibody enzyme-linked immunoassay (ELISA).

ELISA plates were coated overnight at 4°C with 250 ng/well of a goat anti-human Fc fragment. Plates were then washed three times with wash buffer (Dulbecco’s phosphate-buffered saline [DPBS]-containing 0.05% Tween 20) and incubated for 1 h at 37°C with 5% blocking buffer (wash buffer containing 5% nonfat dry milk). After washing the plates three times, serial dilutions (1:5 in 1% blocking buffer) of VRC01 antibody variants starting at 1 μg/mL were added. Plates were further incubated for 1 h at 37°C. Unbound antibodies were removed by three washes, and bound antibodies were detected with a horseradish peroxidase conjugated goat anti-human F(ab′)₂ antibody. After 1 h of incubation at 37°C, plates were washed four times, developed with 3,3′,5,5′-tetramethylbenzidine (TMB) substrate, and stopped with H₂SO₄. Optical densities (at 450 nm) were measured with a Synergy 2 plate reader (BioTek). The IgG concentrations of the VRC01 variants were calculated based on a standard curve generated with a parental VRC01 obtained from the NIH AIDS Reagent Program.