Combining site and antigenic determinants of the V regions

Preparation of Fv fragment and its peptide chain V(L) and V(H) is described. The renaturation of completely unfolded V(L) and V(H) to generate and active Fv indicates that the variable domain, which is only a part of the peptide chains (L or H), contains all the information for its folding and generation of antibody combining site. Model building studies (together with Drs. Davies and Padlan, NIH) were attempted on the basis of known coordinates of other immunoglobulin V regions. The model was found to be compatible with the affinity labeling of protein 315 and with other studies on mapping the combining site. This mapping indicates that Trp 93L is the major residue interaction with the DNP hapten. Indeed, V(L) was found to contain the subsite for the DNP ring and its affinity to DNPOH is similar to that of Fv (V(L)+V(H)). On the other hand DNP haptens with longer side chains bind much better to Fv suggesting that the side chain of the hapten interacts predominantly with V(H). V(L) exists as a dimer which can undergo conformational transition near pH 7. One conformer binds two haptens per dimer, whereas the other binds only one hapten. Thus the combining site made up of two V(L) chains is more flexible than that made up of V(L)+V(H). Antibodies were prepared against V(L) and V(H). These antibodies react with many different V regions on L chains (anti V(L)) or H chains (anti V(H)). Anti V(H) reacts with isolated H chains much better than with intact Ig. These antibodies were used to detect V regions on T cells by employing inhibition of antigen binding T cells. It was found that the inhibition by anti V(H) and anti V(L) affects different T cell populations. Anti-V(H) inhibit the binding to Lyt-1 ⁺ cells known also to be T helper cells, whereas anti-V(L) inhibit antigen binding to Ly-2 ⁺3 ⁺ cells known to be suppressor cells. These results suggest that the expression of V regions on T cells is different from that on B cells. Probably in T cells there is unlinked expression of V(L) and V(H) and they are selectively expressed on different T cell subpopulations.