NEUTRALIZING EPITOPE MAPPING OF 6 BETA(1)-BUNGAROTOXIN MONOCLONAL-ANTIBODIES AND ITS APPLICATION IN BETA(1)-BUNGAROTOXIN PEPTIDE VACCINE DESIGN

Twenty three stable monoclonal antibodies (mAbs) against beta(2)-bunga rotoxin (beta(1)-bgt) were prepared by the hybridoma technique. Seven of the 23 mAbs (mAbs 2, 6, 8, 11, 17, 21 and 22) could inhibit more th an 70 % of phospholipase A(2) activity of beta(1)-bgt and neutralize t he toxin. Six of these neutralizing mAbs (mAbs 2, 6, 8, 17, 21 and 22) recognized continuous epitopes on the A chain of beta(1)-bgt and the other one (mAb 11) recognized a conformational epitope on the toxin. T he continuous epitopes of these six mAbs were mapped using synthetic p eptide and proteolytic enzymes. Experimental results indicate that mAb 17 recognized the A-chain residues 31-37; mAbs 2 and 8 recognized res idues 46-51; mAbs 21 and 22 recognized residues 91-98; and mAb 6 recog nized residue 100-106. The competitive-antibody-binding inhibition exp eriments showed that the affinity of these neutralizing mAbs to the na tive beta(1)-bgt is compatible with synthetic peptides. Furthermore, m ice 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 beta( 1)-bgt challenge. Subsequently, the peptide-immunized sera were passiv ely injected into Balb/c mice and a significantly protective effect wa s also observed. To our knowledge, this study is the first systematic demonstration of multiple neutralizing B-cell epitopes of beta(1)-bgt, and this study is also the first report of the protective synthetic-p eptide vaccine against beta(1)-bgt challenge.