Detection of free and complexed β3 antigen in CHO cells transfected with wild-type or mutant β3 cDNA. A microtiter plate was precoated with 0.6 μg/100 μL of goat antimouse IgG before the addition of either anti-β3 MoAb P37 or anti-αvβ3 MoAb 23C6. The plate was then incubated with transfected CHO cell lysate (50 μg protein). The captured β3 antigen in the wells was detected by the addition of human anti–HPA-1a IgG alloantibodies, and anti–HPA-1a binding was quantitated using peroxidase-labeled goat antihuman IgG according to established ELISA methodology. The optical density (OD) was measured at 490 nm. (A) Quantitation of free versus complexed β3 antigen in CHO cells transfected with β3-wt, β3-met, and β3-pro. (B) Titration curve demonstrating the sensitivity of the ELISA assay with MoAb 23C6 used for the detection of αvβ3 complexes in CHO β3-wt cell lysate.