Serum Antibody Avidity Measurement

Serum binding antibody avidities to HIV-1 Env gp140 were measured by a urea disruption enzyme-linked immunosorbent assay (ELISA) (58 (link)). Briefly, ELISA plates (Thermo Scientific) were coated with individual Env gp140s and incubated overnight. Guinea pig sera were prepared in a dilution plate to values between 1.0 and 1.5 at optical densities of 450 nm (OD₄₅₀) to OD₈₀₀ to provide antibody concentrations within a linear range. Sera were plated in duplicate twice; one duplicate was treated with 8 molar urea (Sigma-Aldrich) and the other with distilled water as a control. Plates were then incubated with an HRP-conjugated goat anti-guinea pig secondary antibody (Jackson ImmunoResearch Laboratories). Plates were developed and read using the SpectraMax plus ELISA plate reader (Molecular Devices) and SoftMax Pro 4.7.1 software (OD₄₅₀ to OD₈₀₀). Percent avidity was calculated using the following formula: [(average absorbance of urea-treated sample/average absorbance of water-treated, matched sample) × 100]. The avidity index describes 0 to 30% as low-avidity, 30 to 50% as moderate-avidity, and >50% as high-avidity binding antibodies.