IgA isolation from cervicovaginal swabs.

To limit the risk of protease digestion of antibodies in the genital secretions, the samples were immediately kept cold and subsequently frozen within a few hours. Any digestion that may have occurred despite these precautions could theoretically have resulted in underestimation of the HIV-neutralizing IgA activity; however, all samples were handled blind to study drug randomization.

Two separate Dacron swabs were used to collect cervical and vaginal mucous samples: one swab was placed into the endocervical opening and gently rotated, and a second Dacron swab was placed along the lateral vaginal mucosal wall and gently rotated. Each swab was placed in a cryovial with 0.5 ml phosphate-buffered saline (PBS) and stored at −80°C. Separate cervical and vaginal aliquots from the same individual were combined prior to IgA isolation. IgA1 (IgA) was purified from thawed samples as previously described (15) with minor modifications. A total of 200 μl of supernatant was diluted 1:4 in PBS (pH 7.4) and added to 400 μl jacalin-agarose beads (ImmunKemi, Stockholm, Sweden). The diluted sample and the jacalin-agarose beads were mixed for 2 h at 4°C followed by centrifugation. Beads were then thoroughly washed with PBS (pH 7.4). The bound IgA was eluted overnight at room temperature by adding 1 ml 0.8 M d-galactose (pH 7.5), and supernatants with purified IgA were thereafter collected without further quantification or normalization. All fractions were stored at −80°C.