Vol. 39 No. 2, 2000

Immunochemical Studies on β₁-Bungarotoxin

Chen-Chung Yang* and Hong-Lin Chan

Department of Life Science, National Tsing Hua University, Hsinchu, Taiwan 300

Chen-Chung Yang and Hong-Lin Chan (2000) β₁-Bungarotoxin (β₁-Bgt) is the main and the most toxic isoform in the β-Bgt family. The toxin consists of 2 dissimilar polypeptide chains: the A chain with 120 amino acid residues and the B chain with 60 amino acid residues, cross-linked by an interchain disulfide bond and possessing weak phospholipase A₂ (PLA₂) activity. The amino acid sequence of the A chain is homologous with those of PLA₂ enzymes from snake venom and the mammalian pancreas, while the B chain serves as a recognition subunit of the toxin towards a specific target presynaptic membrane and blocks the voltage-gated potassium channel. The numbers of antigenic determinants on β₁-Bgt, A chain, and B chain were determined to be 7, 5, and 2, respectively, by the quantitative precipitin reactions and analysis of the molecular weight of the soluble complex formed from β1-Bgt and Fab fragments of the purified antibody. Twenty-three stable monoclonal antibodies (mAbs) were prepared against β₁-Bgt, seven of which could inhibit more than 70% of PLA₂ activity of β₁-Bgt and could neutralize the toxicity of the toxin. Continuous epitopes of β₁-Bgt were mapped, and the result indicates that the A chain sequences 31-37, 46-51, 91-98, and 100-106 are the neutralizing epitopes of β₁-Bgt. A hybrid hybridoma that produces bispecific mAb, which recognizes 2 different epitopes on the A chain of β₁-Bgt at peptide sequences 46-51 and 100-106, has been obtained by fusing 2 hybridoma cell lines. The combination of bispecific mAb, with its 2 corresponding epitopes of β1-Bgt, not only facilitates immuno-complex formation and enhances avidity, but also highly neutralizes the biological activity of β₁-Bgt. Furthermore, mice immunized with BSA-conjugated A-chain-peptide sequences A(31-37), A(46-51), A(91-98), or A(100-106) were protected from a high-dose β₁-Bgt challenge.

Key words: Bungarotoxin, Presynaptic neurotoxin, Monoclonal antibody, Hybrid hybridoma, Bispecific monoclonal antibody, Peptide vaccine.

*Correspondence: -