Combining phage display and molecular modeling to map the epitope of a neutralizing antitoxin antibody

Crotoxin is a potent presynaptic neurotoxin from the venom of the rattlesna ke Crotalus durissus terrificus. It is composed of the noncovalent and syne rgistic association of a weakly toxic phospholipase A(2), CB, and a nontoxi c three-chain subunit, CA, which increases the lethal potency of CB. The A- 56.36 mAb is able to dissociate the crotoxin complex by binding to the CA s ubunit, thereby neutralizing its toxicity. Because A-56.36 and CB show sequ ence homology and both compete for binding to CA, we postulated that A-56.3 6 and CB had overlapping binding sites on CA. By screening random phage-dis played libraries with the mAb, phagotopes bearing the (D/S)GY(A/G) or AAXI consensus motifs were selected. They all bound A-56.36 in ELISA and compete d with CA for mAb binding, although with different reactivities. When mice were immunized with the selected clones, polyclonal sera reacting with CA w ere induced. Interestingly, the raised antibodies retained the crotoxin-dis sociating effect of A-56.36, suggesting that the selected peptides may be u sed to produce neutralizing antibodies. By combining these data with the mo lecular modeling of CA, it appeared that the functional epitope of A-56.36 on CA was conformational, one subregion being discontinuous and correspondi ng to the first family of peptides, the other subregion being continuous an d composed of amino acids of the second family. Phage-displayed peptides co rresponding to fragments of the two identified regions on CA reacted with A -56.36 and with CB. Our data support the hypothesis that A-56.36 and CB int eract with common regions of CA, and highlight residues which are likely to be critical for CA-CB complex formation.