Structural and genetic studies on chicken 7S immunoglobulin allotypes. I. Detection of four heavy chain specificities with a new radioimmunoassay

A method was developed for the detection of allotypes on intact, radiolabeled 7S immunoglobulin (Ig) which does not involve insolubilized alloantisera. Binding of dinitro phenylated anti allotype (DAA) antiserum with radiolabeled alloantigen was demonstrated by the addition of rabbit anti dinitrophenyl antiserum followed by precipitation of complexes with sheep anti rabbit γ globulin antiserum. Dinitrophenylation of alloantiserum up to 8.5 hr had no effect on antibody titer or avidity. Binding inhibition assays with the DAA method detected allotypes on 2 to 5 ng of 7S Ig. Four specificities were detected on 7S Ig heavy (H) chains by binding inhibition with the DAA assay. Specificities CS 1.1 and CS 1.2, located in the Fab fragment, were detected at 3 to 5 fold lower concentrations of reassociated H and light (L) chains than on the isolated H chains. On the basis of regression coefficients calculated from binding inhibition curves, the avidity of isolated H chains was significantly increased following reassociation with L chains, indicating that the conformation of CS 1.1 and CS 1.2 determinants depends in part on H and L chain interactions. In contrast, reassociation of H and L chains did not significantly increase H chain inhibitory activity or avidity for specificities CS 1.3 and CS 1.4.