Immunoprecipitation of the GPIb-IX complex using the rifampicin-dependent antibodies.
After labeling the surface proteins of the platelets with biotin, the GPIb-IX complex was immunoprecipitated using AB serum, patient serum, a nonspecific murine IgG1 (MOPC21), and the GPIX-specific mAb (GRP), all in the presence or absence of the drug as indicated. The platelets were lysed, and the immunoprecipitated proteins were collected using Dynabeads coated with the appropriate capture antibodies. The proteins were analyzed by SDS-PAGE, transferred to PVDF membrane, and detected by streptavidin HRP and chemiluminescence reagents. The GPIb-IX complex (GPIbα, 143-kd band; GPIbβ, 24-kd band; and GPIX, 20-kd band) was only detected with the patient serum in the presence of the drug (lane 4) and with the anti-GPIX mAb (GRP) in both the absence (lane 7) and presence (lane 8) of the drug. Nonspecific bands (165 kd and 65 kd) were seen both with the test, and control antibodies in the presence or absence of the drug and were present in no particular pattern.
