Multiplex Bead Assay for Bloodspot IgG and IgG4

Bloodspot eluates were screened in duplicate with the antigen-coupled beads in a multiplex bead assay [8] (link). Filter-bottom plates (96-well) (Millipore, Bedford, MA) were pre-wet with 0.5% BSA, 0.05% Tween 20, 0.02% sodium azide in PBS (PBN2). Antigen-coupled beads (2500 each) were added to each well and washed twice with PBN2. Control sera and bloodspot eluates (1∶400) were added in duplicate at 50 µl per well to the beads. The plates were vigorously shaken for 30 seconds, covered, and shaken at room temperature for 1.5 hours. After incubation, wells were washed three times with 100 µl of 0.05% Tween 20 in PBS (PBST) with a vacuum device (Millipore). Total IgG was detected with 50 ng of biotinylated mouse anti-human total IgG (clone H2; Southern Biotech, Birmingham, AL) and 40 ng of biotinylated mouse anti-human IgG4 (clone HP6025; Invitrogen, South San Francisco, CA) per well in 50 µl PBN2. After incubation, wells were washed as above. R-phycoerythrin-labeled streptavidin (Invitrogen, South San Francisco, CA) was added at a concentration of 250 ng per well and incubated for 30 minutes at room temperature. Wells were washed as previously after incubation. Wells were additionally incubated in 50 µl of PBN2 to remove any loosely bound antibodies for 30 minutes with shaking. After the final incubation in PBN2, wells were vacuum-evacuated and washed once with PBST. Beads were suspended in 125 µl PBS, shaken, and immediately read on a BioPlex 200 instrument (Bio-Rad, Hercules, CA) equipped with Bio-Plex Manager 6.0 software (Bio-Rad).