Demonstration of two distinct antigenic determinants on human alpha-foetoprotein by monoclonal antibodies.

Mice were immunized with a purified preparation of human alpha-foetoprotein (AFP) and their spleen cells were hybridized with mouse myeloma cells. Eleven hybridoma cell lines secreting antibody to AFP were obtained. These antibodies were classified into two groups on the basis of different antigenic determinants they recognized. Seven cell lines produced antibodies directed to one determinant of AFP (determinant a), while the remaining 4 produced antibodies to another determinant (determinant b). A native AFP molecule bore one each of a and b determinants which were accessible by monoclonal antibodies; it was detected by a sandwich-type solid-phase radioimmunoassay only when antibody to a was used for coating wells of the microtitre plate and antibody to b as radiolabelled reagent, or vice versa. The presence of two different antigenic determinants on AFP was further confirmed by a chemical modification. When AFP was reduced in the presence of 2 mM dithiothreitol and then alkylated, determinant b was completely destroyed, but the determinant a remained unaffected. Furthermore, reduced and alkylated AFP was detected by the radioimmunoassay employing antibody to a both for coating wells and as radiolabelled reagent, thereby indicating that it bore two determinant as, one of which had been unaccessible in the native AFP.