Antibodies against a synthetic peptide designed to mimic a surface area of the H chain of botulinum neurotoxin A

Abstract

A surface-simulation peptide, SQMIN[GG]TTNI[G]NSIS[G]RDTH[G]NLES, (SS-peptide) was synthesized that described the spatial interrelationships of 21 residues on the surface of botulinum neurotoxin type A (BoNT/A). The glycine residues in brackets were spacers between surface segments of BoNT/A. The SS-peptide did not contain an antigenic or a synaptosome (snps)-binding site of BoNT/A and it did not bind anti-BoNT/A antibodies (Abs) or inhibit toxin binding to synaptosomes. Antibodies prepared by immunization with the free peptide or with peptide-ovalbumin (OVA) conjugate did not protect mice in vivo against a lethal dose of the toxin. Early Abs (day 52) against free SS-peptide recognized the peptide and showed a small cross-reaction with native toxin, but later Abs (day 115) exhibited a higher cross-reaction with to active toxin. Similarly, early Abs (day 52) against peptide-OVA conjugate displayed a low cross-reaction with native toxin, but the cross-reaction also increased in later bleeds (day 115). Both, the free peptide or its OVA conjugate, elicited predominantly IgG Abs that in the course of immunization were increasingly more capable of binding to a peptide conformation resembling the shape of the surface area on the native BoNT/A. The Abs were able to detect the conformational changes of the toxoid. This demonstrates that Abs could be prepared essentially against a peptide that mimics a surface area and such Abs could recognize and bind to the correlate surface area on the native protein. The area selected could, but need not, be an antigenic site when the native protein is used as an immunogen. The ability to make Abs against protein surface areas that are mimicked by surface-simulation synthesis provides versatile and valuable tools for analytical, therapeutic, clinical and diagnostic applications.