Multiscreen bv

Immediately before use, the antibody-coupled beads were vortexed and 2x10³ beads were dispensed in pre-wet wells of 96-well filter bottom plates (Multiscreen BV; Millipore, Billerica, MA). Assays were run either in monoplex or multiplex format. Antibody-coupled beads were incubated with serially diluted extracts for one hour in the dark at room temperature with gentle mixing. The bound allergens were detected with a mixture of rabbit antisera containing anti-E6Cg, anti-Bla g 1, anti-Bla g 2, and anti-Bla g 4 (each diluted 1:500). Biotin-coupled anti-rabbit IgG antibody (KPL, Gaithersburg, MD) was diluted (1:1000) and added to each well for detection. The binding was detected using diluted streptavidin-R-Phycoerythrin (100 μg/mL) (Thermo Scientific). After the final incubation the beads were resuspended in sheath fluid (Bio-Rad Laboratories) and median fluorescence intensities (MFI) were measured in a BioPlex 200 reader (Bio-Rad Laboratories).

The bead based multiplex suspension assay was performed as described by Khurana et al., [22 ]. Briefly 18 different scFv antibody-coupled beads were mixed and dispensed in pre-wet wells of 96-well filter bottom plates (Multiscreen BV; Millipore, Billerica, MA) containing serially diluted termite extract. Antibody-coupled beads were incubated with termite extract for 1 hr in the dark at room temperature with gentle mixing. The binding of scFv and proteins was detected with a mixture of rabbit antisera containing anti-E6Cg, anti-Bla g 1, anti-Bla g 2, and anti-Bla g 4 (each diluted 1:500) [23 (link)]. Biotin-coupled anti-rabbit IgG antibody (KPL, Gaithersburg, MD) was diluted (1:1000) and added to each well for detection. The binding was detected using diluted streptavidin-R-Phycoerythrin (100 μg/mL) (Thermo Scientific). Finally, the beads were resuspended in sheath fluid (Bio-Rad Laboratories) and median fluorescence intensities (MFI) were measured in a BioPlex 200 reader (Bio-Rad Laboratories).

The MAP assay was performed as previously described [16 (link), 17 (link), 27 (link)]. In brief, 50 µl of each antigen pool (containing 2000 microspheres of each antigen) were incubated with 50 µl of a 1:200 dilution of plasma in PBS-1% BSA in pre-wetted filter plates (96 well Multiscreen BV; Millipore) for 1 h at 25 °C on a rotating shaker at 650 rpm. Microspheres were washed twice with PBS-0.05% Tween20 and once with PBS-1% BSA. Then, 100 µl of secondary Ab [R-phycoerythrin-conjugated, Affini Pure F(ab′)₂ fragment, goat anti-human IgG Fc fragment-specific, Jackson Immunoresearch] diluted to 2 µg/ml in PBS-1% BSA was added to each well and incubated as above in the dark for 1 h. Microspheres were washed as described above, re-suspended in 100 µl PBS-1% BSA, and the microsphere suspension was analysed using a Liquichip M100 reader (Luminex Corp., Austin, TX). The reader was programmed to read a minimum 100 beads per spectral address, DD Gate 7500–15,000 and 35 s timeout. The results were expressed as median fluorescence intensity (MFI). The serological assays were repeated twice and the average of the two MFIs was used for analysis.