Preparation of monoclonal antibodies to murine platelet glycoprotein IIb/IIIa (alpha IIb beta 3) and other proteins from hamster-mouse interspecies hybridomas

To obtain mouse-specific monoclonal antibodies (mAbs) against platelet prot eins, 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 ut ilizing an ELISA in which the target antigen was mouse platelets adsorbed o nto microtiter plates in the presence of thrombin, Secondary screening assa ys included ELISA tests using murine fibrinogen or platelets from beta 3-in tegrin knockout mice, how cytometry, immunoblotting, immunoprecipitation, a nd a functional assay to identify antibodies that inhibit platelet-fibrinog en interactions. Hybridoma cells producing hamster mAbs against murine glyc oprotein (GP) IIb/IIIa, fibrinogen, CD9, and other platelet integrins were identified. Two hybridomas (1B5 and 9C2) producing antibodies that react wi th the GPIIb/IIIa complex in immunoprecipitation analysis were subcloned tw ice. Functional analyses by means of aggregation and adhesion assays reveal ed that 1B5 completely inhibits platelet-fibrinogen interactions, whereas 9 C2 does not affect platelet aggregation or platelet adhesion. (C) 1999 Acad emic Press.