The antigen-binding domain of a human IgG-anti-F(ab')2 autoantibody

Abstract

Recent studies revealed an immunoregulatory role of natural IgG-anti-F(ab')2 antibodies in both healthy individuals and patients with certain diseases. The implication of anti-F(ab')2 antibodies in the pathogenesis of diseases prompted us to study the gene segment structure of their antigen-binding domains and their binding characteristics. cDNA 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 amplified by PCR and inserted into a phagemid surface expression vector. Single-chain antibodies displayed on the phage surface were screened for binding to F(ab')2 fragments. The subsequent analysis of 95 single clones demonstrated that they all bound specifically to F(ab')2. Sequence analyses of 12 clones showed that 11 were identical and 1 contained 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 and the light chains to the V(kappa)2 gene family. The 11 identical light-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 relatively high affinity (Ka = 2.8 x 10(7) M(-1)). The strong binding capacity was further demonstrated by competitive inhibition of the serum anti-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 clones and describes the binding kinetics of the purified monomeric fragments.

Figure 1

Binding of scFv anti-F(ab′)₂-expressing phages to F(ab′)₂ fragments. (A) The polyclonal phage population obtained after each selection round was tested in ELISA for binding to F(ab′)₂. Negative controls consisted of microtiter plates coated with blocking buffer or BSA. (B) Single clones (n = 95) with anti-F(ab′)₂ activity were generated and tested in ELISA (negative controls: A₆₂₀ of blocking buffer = 0.079 and that of BSA = 0.074).

Figure 2

Sequence analysis of V_H and V_L chain genes of scFv anti-F(ab′)₂ antibodies. Amino acid sequences are shown. FR, framework region; CDR, complementarity-determining region; CONST., constant region.

Figure 3

Binding of scFv anti-F(ab′)₂ antibodies to different antigens [F(ab′)₂, Fab, Fc, IgG, and BSA] is analyzed by ELISA.

Figure 4

Comparison of BIAcore sensorgrams for the interaction between recombinant antibody fragments and immobilized antigens. Overlap plots showing interaction of scFv2 (A) and scFv6 (B) with immobilized F(ab′)₂ at different scFv concentrations. Interaction of scFv2 (C) and scFv6 (D) at 400 nM with different antigens. The association and dissociation phases of the sensorgrams are shown. The bulk effect is subtracted from the relative response. Legends in the right of each plot show the list of scFv concentrations (A and B) or immobilized antigens (C and D) in the order of decreasing resonance signals. RU, response units.

Figure 5

Competitive inhibition of serum IgG-anti-F(ab′)₂ antibodies with scFv anti-F(ab′)₂ antibodies. ELISA plates coated with F(ab′)₂ γ-fragments were incubated with increasing dilutions of scFv2 (A), scFv6 (B), and a human anti-digoxigenin scFv antibody (C). Serum IgG-anti-F(ab′)₂ antibodies were added at a dilution of 1:512. Negative controls consisted of PBS. Maximum binding activity of scFv2 and scFv6 was A₆₂₀ = 0.983 and 0.869, respectively.