Quantitation of HIV-1-Specific IgG, IgA, and IgM Antibodies in Human Genital Tract Secretions

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Abstract
A quantitative enzyme-linked immunosorbent assay (ELISA) has been developed for the quantitation of HIV-1-specific immunoglobulins of the IgG, IgA. and IgM isotypes and was used to assess HIV-specific antibody concentrations in semen and cervicovaginal lavage (CVL) specimens. Immunoglobulin isotype concentrations were assessed by capture ELISA using immunoglobulin isotype-specific secondary antibodies and commercial IgG. IgA, and IgM standards. HIV-1 antibody detection kits (Abbott Laboratories, North Chicago, IL, U.S.A.) and immunoglobulin isotype-specific secondary antibodies were used to obtain optical density (OD) units for HIV-1-specific antibodies of each isotype. To determine the antibody concentrations from the OD values, ODs were compared with those from HIV-1-specific antibody isotype standards of known concentration obtained from CVL specimens of seropositive women by affinity binding to HIV antigen-coated beads and acid elution. The sensitivity of the HIV-1-specific immunoglobulin assay was 0.01 µg/ml for IgG, 0.04 µg/ml for IgA. and 0.08 µg/ml for IgM. The interassay coefficient of variation for the different immunoglobulin isotypes varied from 5% to 33%, being lowest for IgG and highest for IgM. HIV-1-specific antibodies were detected in all semen samples from seropositive men in concentrations ranging from 53 to 261µg/ml. Thirteen of 14 samples contained high levels of HIV-1-specific IgG antibodies (22-72µg/ml) whereas 10 of the 14 (71%) semen samples contained detectable but lower levels of HIV-1-specific IgA and IgM (maximum level: 3.7 µg/ml for IgA and 14.8µg/ml for IgM). HIV-1-specific IgG antibodies were detected in all 196 CVL samples from seropositive women in concentrations ranging from 0.01 to 47 µg/ml, whereas only 16 women (8%) had IgA levels above the detectable limit (range, 1.4-3.9µg/ml), and only eight women (4%) had IgM levels above the detectable limit (range, 1.8-8.6µg/ml). None of 80 low-risk women or 20 low-risk men (negative controls) had detectable levels of HIV-1-specific antibodies in genital tract specimens. HIV-1-specific IgG levels in CVL specimens of seropositive women were significantly higher in individuals who had acquired HIV through heterosexual transmission, and a majority of women with elevated levels of HIV-specific IgA isotype antibodies in CVL samples had evidence of genital tract inflammation (>104 polymorphonuclear leukocytes[PMNs]/ml). Use of this quantitative method will facilitate direct comparison of data obtained within and among laboratories and enable further research on factors affecting antibody levels in genital tract secretions and their effects on HIV-1 transmission.