Abstract
β 1 -Bungarotoxin (β 1 Bgt) is the main and the most toxic isoform 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 disultide bond and possessing weak phospholipase A 2 (PLA 2 ) activity. The amino acid sequence of the A chain is homologous with those of PLA 2 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 β 1 -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 β 1 -Bgt and Fab fragments of the purified antibody. Twenty-three stable monoclonal antibodies (mAbs) were prepared against β 1 -Bgt, seven of which could inhibit more than 70% of PLA 2 activity of β 1 -Bgt and could neutralize the toxicity of the toxin. Continuous epitopes of β 1 -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 β 1 -Bgt. A hybrid hybridoma that produces bispecific mAb, which recognizes 2 different epitopes on the A chain of β 1 -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 β 1 -Bgt, not only facilitates immuno-complex formation and enhances avidity, but also highly neutralizes the biological activity of β 1 -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 β 1 -Bgt challenge.