R phycoerythrin conjugated streptavidin

A capture multiplex assay was performed to determine competing binding of the Pentamer-specific monoclonal antibodies. For direct competition between pairs of monoclonal antibodies Luminex microspheres (MagPlex microspheres, Luminex Corp. cat# MC100XX), of different classification, were coupled with individual monoclonal antibodies by chemical coupling according to manufacturer instructions. Individual antibodies were also biotinylated using a commercial kit (NHS-PEG4-Biotin, No-Weig Format, Thermo Scientific, cat# 21329). In 96 well white plates, 1000 microspheres/well for each classification/monoclonal were mixed in 50 μl/well of DPBS + 1% BSA + 0.05% sodium azide (assay buffer) with purified Pentamer, starting at 100 ng/well with three-fold dilutions down to 0.05 ng/well. After washing three times with 200 μl/well of PBS containing 0.05% (w/v) Tween 20 (wash buffer) to remove excess antigen, individual biotinylated monoclonal antibodies (at 0.75–1.5 μg/ml) were added in separate wells in 50 μl/well of assay buffer and incubated for 1 h at RT with orbital shaking in the dark. After washing, R-Phycoerythrin conjugated Streptavidin (Jackson ImmunoResearch, cat# 016-110-084) was added, 50 μl/well in assay buffer, and incubated for 1 h at RT. After a final wash, Fluorescence intensity was measured using a Luminex FlexMap 3D (Life Technologies model FM3D000).