Biotinylated mouse antihuman total igg clone h2

The DBS were shipped to the CDC in the United States at ambient temperature for testing by serologic assays for antibodies to the C. trachomatis antigens Pgp3 and CT694. Antigen-coupled beads were added to 96-well plates (Millipore, Bedford, MA). Control sera and blood spot eluates (1:400) were then added to appropriate wells, beads suspended, plates covered, and shaken at room temperature for 1.5 hours. After washing the beads, total IgG was detected using biotinylated mouse antihuman total IgG (clone H2; Southern Biotech, Birmingham, AL) and biotinylated mouse antihuman IgG4 (clone HP6025; Invitrogen, South San Francisco, CA). After a second wash, streptavidin-phycoerythrin (SAPE Invitrogen) was added at a concentration of 250 ng per well and incubated for 30 minutes at room temperature. Beads were read on a Luminex instrument (Luminex Corp., Austin, TX) equipped with Bio-Plex Manager 6.0 software (Bio-Rad, Hercules, CA). The signal was converted to median fluorescence intensity (MFI) with background levels subtracted out (MFI-BG). Positivity thresholds were generated using a receiver-operating characteristic curve panel of specimens of previously classified positive or negative samples.^{5 (link),7 (link)} The positive thresholds for Pgp3 and CT694 were 1558 MFI-BG and 164 MFI-BG, respectively.