The antigen binding domain of non-idiotypic human anti-F(ab ')(2) autoantibodies: Study of their interaction with IgG hinge region epitopes

In previous studies we described a natural human IgG-anti-F(ab')(2) autoant ibody family with immunoregulatory properties, Genes coding for the variabl e regions of the heavy and light chains of the Abs were isolated from a nat ural Ig gene library and scFv Abs were expressed in E, coli. The scFv Abs b ound to F(ab')(2) but not to Fab fragments. This points to an epitope locat ed in the hinge region since Fab fragments are lacking most of the hinge. I n order to verify our hypothesis, double chain peptides comprising the lowe r-, middle-, and part of the upper hinge subregion of IgG1-IgG4 were synthe sized on cellulose membranes and tested for binding to the Abs. The results show binding of Abs to IgG1 and IgG4 hinge region peptides, In order to id entify the key residues of the discontinuous epitopes we carried out comple te substitutional analyses in which each amino acid of the wt peptides was substituted by all other amino acids except cysteine. The exchange of proli ne in the IgG1 or IgG4 middle hinge region abrogated the binding, revealing the importance of this subregion for epitope expression. No binding to the IgG2 or IgG3 hinge was detected. These results indicate that scFv anti-F(a b')(2) Abs recognize the hinge region of IgG1 and IgG4 and that the express ion of the epitope depends on an intact middle hinge subregion. Human Immun ology 60, 282-290 (1999). (C) American Society for Histocompatibility and I mmunogenetics, 1999, Published by Elsevier Science Inc.