Investigation of human platelet alloantigens and glycoproteins using non-radioactive immunoprecipitation

Sensitive techniques to detect platelet antibodies are needed for the investigation of immune thrombocytopenic syndromes such as neonatal alloimmune thrombocytopenia and post-transfusion purpura. Radioimmunoprecipitation has proved useful in the investigation of platelet-antibody interactions; however, the requirement for a radioactive label is a disadvantage. We describe the immunoprecipitation of human platelet proteins labelled with nonradioactive NHSS-biotin and compare the results with proteins labelled with 125I. The efficiency of labelling was evaluated by immunoprecipitation using well-characterized human anti-platelet antisera and murine monoclonal antibodies. The immunoprecipitated proteins were separated by SDS-PAGE, transferred to nitrocellulose and detected using streptavidin-horseradish peroxidase and a chemiluminescent substrate with exposure to X ray film. The biotinylation technique labelled glycoproteins Ia/IIa, Ib/IX, IIb/IIIa, IV, and p175 which carry all of the known platelet alloantigens and isoantigens. It was as sensitive as radiolabelling and had the advantage of labelling GPs Ib beta and IX, which were not labelled using radioiodine. Human sera containing alloantibodies to HPA-1a on GP IIIa, HPA-3a on GP IIb, HPA-5a and HPA-5b on GP Ia, Govb on p175, and the isoantibody Naka on GP IV precipitated the corresponding biotinylated proteins. Biotinylated proteins could be detected using a 30 s exposure compared to 2 days or longer for 125I. Immunoprecipitation of human platelet glycoproteins labeled with NHSS-biotin is a fast and sensitive alternative to conventional radioimmunoprecipitation for the study of human platelet antigens.