THE ANTIGEN-BINDING DOMAIN OF A HUMAN IGG-ANTI-F(AB')(2) AUTOANTIBODY

Recent studies revealed an immunoregulatory role of natural IgG-anti-F (ab')(2) antibodies in both healthy individuals and patients with cert ain diseases, The implication of anti-F(ab')(2) antibodies in the path ogenesis of diseases prompted us to study the gene segment structure o f their antigen-binding domains and their binding characteristics. cDN A was prepared from the lymphocytes of a patient with a high IgG-anti- F(ab')(2) serum titer, Variable heavy and light gene segments were amp lified by PCR and inserted into a phagemid surface expression vector, Single-chain antibodies displayed on the phage surface were screened f or binding to F(ab')(2) fragments, The subsequent analysis of 95 singl e clones demonstrated that they all bound specifically to F(ab')(2), S equence analyses of 12 clones showed that 11 were identical and 1 cont ained a silent point mutation in the heavy chain and three amino acid exchanges in the light chain, The heavy chains belonged to the V(H)3 a nd the light chains to the V(kappa)2 gene family, The 11 identical lig ht-chain genes were completely homologous to a germ-line sequence (DPK -15), Binding assays showed that the single-chain antibodies bind to F (ab')(2), but not to Fab, Fc, or intact IgG, This binding pattern was confirmed by surface plasmon resonance studies, which revealed a relat ively high affinity (K-a = 2.8 x 10(7) M(-1)). The strong binding capa city was further demonstrated by competitive inhibition of the serum a nti-IgG antibody's interaction with antigen, The present study defines for the first time to our knowledge the gene segment structure of the antigen-binding domain of two human IgG-anti-F(ab')(2) autoantibody c lones and describes the binding kinetics of the purified monomeric fra gments.