Development and standardization of methods to evaluate the antibody response to an HIV-1 candidate vaccine in secretions and sera of seronegative vaccine recipients

Enzyme-linked immunosorbent assays (ELISA) were developed to test, in serum and mucosal samples, total IgG, total IgA, serum albumin, and anti-gp120 MN and anti-p24 LAI IgG and IgA levels. These ELISAs were optimized according to reagents and experimental conditions. Inter- and intra-assay coefficients of variation ranged from 3.3% to 18.6%. The ELISA results were linear and precise, and for anti-HIV-1 IgG and IgA, the analytical recovery was close to 100%. For IgG and IgA titration against gp120 MN and p24 LAI, standards were made using pooled sera or gammaglobulins with assigned titres in ELISA units per ml (EU/ml). These standards were used to obtain a linear regression curve that could then be used to obtain the titres of experimental samples. The cut-offs for positivity were determined for sera and mucosal fluid using healthy controls. Validation conditions were defined for ELISAs, and samples that did not satisfy these conditions were retested. Measurement of total IgG and IgA allowed normalization and comparison of the results of specific immunoglobulin levels between different samples. Serum albumin was tested as a marker of transudation from serum to mucosal fluid, allowing calculation of the relative coefficient of excretion, which is one element required to determine the origin of the immunoglobulin detected in mucosal samples. These ELISAs were developed with samples from HIV-1-infected and healthy subjects. We now have the tools to study and understand mucosal immunity in seronegative subjects vaccinated with an HIV-1 candidate vaccine.