Identification of Rat Erythrocyte Antigens with a New Non-Radioactive Immunoprecipitation Technique

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Abstract
In order to examine how rat erythrocytes stimulate eryihrroyte autoantibody production at the molceular level. we have identified rat erythrocyte antigens by immunoprecipition and western blotting using monoclonal antibodies and antisera A novel non radioactive immunoprecipitation technique was used, which employed biotin as a label and a luminescent detection system. The new method was validated by comparison with conventional immunoprecipitation using125I Glyeophorins of relative molecular mass (M1, 81,000 and 38,000 were found to be the major antigenic components of rat erythrocytes. while band 3(the most abundant erythrocyte membrane protein) was not retognized by rat-specific antibodies. The same surface antigens were recognized by sera from mice producing erythrocyte autoantibodies and by sera from mice in which autoantibody production was suppressed. Nine other minor rat-specific antifiens were identified by blotting. ranging in M1 from 23,000 to 147,000, Analysis of the integral membrane proteins of rat and mouse erythrocytes by sodium dodecyl sulphate (SDS) electrophoresis followed by silver or periodic acid-Schiff (PAS) stains revealed differences beween the glyeophorins, but not between rat and mouse band 3. Thus, the major antigenic differences correspond to discernible biochemical differences between rat and mouse erthrocyte sialoglycuproteins.