Light chain of natural antibody plays a dominant role in protein antigen binding

Examinations of the contribution and the specificity of heavy (H) and light (L) chains of natural antibodies to antigen binding may help us to better understand antigen recognition and the development of naive B cells. We pre viously generated natural Fab antibody fragments reactive to preS1 of HBV u sing a naive, non-immunized Fab antibody library derived from peripheral B cells of a normal healthy volunteer. We now constructed expression vectors for the Fd (VH + CH1), L chain, and scFv fragments using the sequences enco ding parental Fabs as a source of natural antibody genes. The recombinant a ntibody fragments were expressed as inclusion bodies in E. coli BL21 (DE) c ells. When denatured and then refolded, the antibody fragments retained the ir binding properties. Recombinant L chains and scFvs exhibited three- to 4 0-fold higher affinities (in the order of 107 M-1) over the parental Fabs, whereas the affinities of Fds (in the order of 10(5) M-1) were much lower c ompared to the parental Fabs. The results obtained from sandwich ELISA reve aled that the L chains bound the virus more efficiently than Fds. Additiona l experiments were performed to evaluate the specificity of the recombinant fragments for surface proteins of HBV. Fds and L chains were reactive towa rds HBsAg and the preS2 peptide as well as preS1 and showed patterns of epi tope recognition quite different from those of parental Fabs. The data pres ented here demonstrate that the prominence of the L chain in determining pr otein binding activity is a property of natural antibodies and is quite unl ike the antibodies induced by immunization, and that the specificity of Fab is not determined by the individual antibody chain but by the correct pair ing of H and L chain. (C) 2000 Academic Press.