Synergy test for recognition of epitopes on soluble proteins; its application in the study of CD21 and CD23 antigens and their respective antibodies

Abstract

Antigens such as CD21 and CD23, which express only one copy of an epitope require two monoclonal antibodies (mAbs) for their detection and estimation. This requirement is exploited in two ways in a technique based on the chromic chloride haemagglutination test. For simple titration of antigen two portions of red cells each coated with one of a pair of synergising mAbs are used in a 1:1 combination. For testing the antigenic specificity of a mAb and assessing its region of epitope binding, the mAb under test is serially diluted in fluid containing a standard amount of antigen and red cells are added to which have been attached a different mAb. If the red cell-bound mAb recognises a determinant topographically distinct from that of the soluble mAb, red cell agglutination to high titre occurs. In titrations of ascitic fluid containing approximately 1 mg/ml mAb, titres of log 29 to log 216 were recorded from a starting dilution of 1 in 200. Hence the test is very sensitive and only minute amounts of a mAb are required for testing. The same test system can be used for assessing the relative display of epitopes on antigen obtained from different sources, e.g., culture supernates and body fluids. The method is of general applicability to monomeric antigens and its use is illustrated by analysis of CD21 and CD23 antigens and antibodies.