Anti human iga hrp

Gp120-specific IgG concentration and gp120-specific IgA titers were determined by ELISA as described before (Ruiz et al., 2016 (link)). 96-well flat-bottomed half-area plates (GreinerBio-One, Germany) were coated with 25 ng/well of gp120_BAL. For gp120-specific IgG quantitation, 25 μl/well of 1/10000 (C, CT and HIV⁺-P) or 1/10 (ESN and HD) plasma dilutions were dispensed in triplicate. A standard curve was constructed, consisting in two-fold serial dilutions of the anti-gp120 2G12 monoclonal antibody starting at 24 ng/ml. IgG detection was performed using an anti-human IgG antibody labeled with horseradish peroxidase (HRP, Sigma-Aldrich, USA) and developed with TMB-ELISA solution (BD Biosciences, USA). Absorption at 450 nm was read on Multiskan EX Microplate Reader (Thermo/Labsystems). IgG concentration was extrapolated from the standard curve and multiplied by the dilution factor. Gp120-specific IgA levels were determined by end-point titration. Two-fold serial dilutions of plasma were prepared, starting at 1:20. Secondary antibody was an anti-human IgA-HRP (Sigma-Aldrich, USA). Plates were developed and read as described for IgG. End-point IgA titer was defined as the reciprocal of the highest plasma dilution at which the average OD value was ≥ 2-fold the average OD value of control wells. Sera from HIV-negative subjects were tested as controls.