To obtain mouse-specific monoclonal antibodies (mAbs) against platelet proteins, an Armenian hamster was immunized with washed mouse platelets. Immune splenocytes were then fused with a nonsecreting murine myeloma cell line, and the resulting heterohybridomas were screened for antibody production utilizing an ELISA in which the target antigen was mouse platelets adsorbed onto microtiter plates in the presence of thrombin. Secondary screening assays included ELISA tests using murine fibrinogen or platelets from beta3-integrin knockout mice, flow cytometry, immunoblotting, immunoprecipitation, and a functional assay to identify antibodies that inhibit platelet-fibrinogen interactions. Hybridoma cells producing hamster mAbs against murine glycoprotein (GP) IIb/IIIa, fibrinogen, CD9, and other platelet integrins were identified. Two hybridomas (1B5 and 9C2) producing antibodies that react with the GPIIb/IIIa complex in immunoprecipitation analysis were subcloned twice. Functional analyses by means of aggregation and adhesion assays revealed that 1B5 completely inhibits platelet-fibrinogen interactions, whereas 9C2 does not affect platelet aggregation or platelet adhesion.
Copyright 1999 Academic Press.