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 previously generated natural Fab antibody fragments reactive to preS1 of HBV using 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 encoding parental Fabs as a source of natural antibody genes. The recombinant antibody fragments were expressed as inclusion bodies in E. coli BL21 (DE) cells. When denatured and then refolded, the antibody fragments retained their binding properties. Recombinant L chains and scFvs exhibited three- to 40-fold higher affinities (in the order of 10(7) M(-1)) over the parental Fabs, whereas the affinities of Fds (in the order of 10(5) M(-1)) were much lower compared to the parental Fabs. The results obtained from sandwich ELISA revealed that the L chains bound the virus more efficiently than Fds. Additional experiments were performed to evaluate the specificity of the recombinant fragments for surface proteins of HBV. Fds and L chains were reactive towards HBsAg and the preS2 peptide as well as preS1 and showed patterns of epitope recognition quite different from those of parental Fabs. The data presented here demonstrate that the prominence of the L chain in determining protein binding activity is a property of natural antibodies and is quite unlike the antibodies induced by immunization, and that the specificity of Fab is not determined by the individual antibody chain but by the correct pairing of H and L chain.
Song
M K
MK
Department of Microbiology, Inje University, Pusan, 614-735, Korea.
Oh
M S
MS
Lee
J H
JH
Lee
J N
JN
Chung
J H
JH
Park
S G
SG
Choi
I H
IH
D000915:Antibody Affinity; D000918:Antibody Specificity; D000936:Antigen-Antibody Complex / Q000737:chemistry; D000937:Antigen-Antibody Reactions / Q000276:immunology*; D004926:Escherichia coli; D006801:Humans; D007147:Immunoglobulin Light Chains / Q000235:genetics / Q000276:immunology*; D011487:Protein Conformation
D000936:Antigen-Antibody Complex; D007147:Immunoglobulin Light Chains