In vitro binding assays in solid phase and in solution. (A) Saturation binding of biotinylated PF4 (biot-PF4) to β2GPI; Immunoplates coated with a constant concentration (10 μg/mL) of recombinant β2GPI or β-lactoglobulin were incubated with various concentrations (0-5 μg/mL) of biot-PF4. After the addition of AP-streptavidin, the OD was measured at 410 nm. Values are the mean ± SD. (B) Homologous inhibition of biot-PF4 binding to β2GPI-immobilized onto microtiter plates, after preincubation with recombinant β2GPI. Preincubated biot-PF4 (1 μg/mL) with increasing concentrations of soluble β2GPI or β-lactoglobulin (0-13.5 μg/mL) for 30 minutes at 37°C was added to immobilized β2GPI onto microtiter plates and incubated overnight at 4°C. After AP-streptavidin and pNPP substrate, OD values were measured at 410 nm and converted to percentage of inhibition. (C) Precipitation with NeutrAvidin-agarose beads and Wester blot analysis with monoclonal anti-PF4 antibody. Biotinylated recombinant β2GPI (biot-β2GPI) and nonbiotinylated β2GPI (β2GPI), used as negative control, were incubated with PF4 in equal molarities (0.1mM and 0.2mM). After incubation, avidin-agarose beads were added to the mixtures, and the resin-bound complexes were eluted with boiling. Eluted proteins were analyzed by SDS-PAGE and were immunoblotted with monoclonal anti-PF4. MW indicates molecular weight marker; lane 1, eluent from biot-β2GPI–PF4–resin sample (0.2mM); lane 2, eluent from biot-β2GPI–PF4–resin sample (0.1mM); lane 3 (negative control), eluent from β2GPI–PF4–resin sample (0.2mM); lane 4, recombinant PF4.