A procedure is described for rapid concurrent synthesis on solid supports of hundreds of peptides, of sufficient purity to react in an enzyme-linked immunosorbent assay. Interaction of synthesized peptides with antibodies is then easily detected without removing them from the support. In this manner an immunogenic epitope of the immunologically important coat protein of foot-and-mouth disease virus (type O1) is located with a resolution of seven amino acids, corresponding to amino acids 146-152 of that protein. Then, a complete replacement set of peptides in which all 20 amino acids were substituted in turn at every position within the epitope was synthesized, and the particular amino acids conferring specificity for the reaction with antibody were determined. It was found that the leucine residues at positions 148 and 151 were essential for reaction with antisera raised against intact virus. A lesser contribution was derived from the glutamine and alanine residues at positions 149 and 152, respectively. Aside from the practical significance for locating and examining epitopes at high resolution, these findings may lead to better understanding of the basis of antigen-antibody interaction and antibody specificity. Recombinant DNA technology now makes possible by deduction from the determined nucleotide sequences reliable amino acid sequences of biologically important proteins. However, methods for identifying the loci in a protein that constitute the antigenic and immunogenic epitopes are few and time consuming and form the bottleneck to further rapid progress. Immunogenic epitopes are defined as those parts of a protein that elicit the antibody response when the whole protein is the immunogen. These immunogenic epitopes are believed to be confined to a few loci on the molecule (1-3). On the other hand, a region of a protein molecule to which an antibody can bind is defined as an antigenic epitope. Antisera prepared against chemically synthesized peptides corresponding to short linear tracts of the total polypeptide sequence have been shown to react well with the native protein (4-9). However, interactions were also found to occur even when the site of interaction did not correspond to an immunogenic epitope of the native protein. This has been interpreted to mean that the number of immunogenic epitopes of a protein is less than the number of antigenic epitopes (4). Conversely, since antibodies produced against the native protein are, by definition, directed to the immunogenic epitopes, it follows that peptides reacting with these antibodies must contain elements of the epitopes. From a study of the few proteins for which the determinants have been accurately mapped, it is postulated that a determinant may consist of a single element (continuous) or of more than one element brought together from linearly distant regions of the polypeptide chain by the folding of that chain as it exists in the native state (discontinuous) (10). Systematic mapping of all the detectable reactive elements of a protein by the ...

Antisera were raised against the chemically synthesized peptide corresponding to each epitope of three foot-and-mouth disease virus strains. Peptide synthesis was further used to determine which amino acid residues in each epitope are important for the specificity of antisera raised against the whole virus. The specificity of the antibody paratope for its epitope was shown to depend on structure as well as sequence. Anti-virus sera demonstrated a greater specificity for the homologous peptide than did the anti-peptide sera. Two of the three peptides were able to induce neutralizing antibodies against the homologous virus. The specificities of the antibodies present in the anti-peptide sera were also inferred from the reactions of each with related sets of peptides. The cross-reactions observed for the anti-peptide sera were readily explained in terms of the antibody specificities determined to be present. The findings also suggest that the diversity of antibodies raised against small peptides is limited and is determined by the immune system. A similar limited response to the native protein was observed, which may account for the high frequency with which anti-peptide sera react with the native homologous protein.Antibodies to protein epitopes are usually highly specific and able to distinguish between proteins differing by only a single amino acid in the region of antibody binding (1-3). Structural changes induced by denaturation or chemical modification of a protein may result in the complete loss of binding by antisera against the native conformation (4, 5). These observations have led to the conclusion that antibody specificity is determined by both the sequence of amino acids and the conformation at the region of binding. In contrast to the relatively stable structure of a protein in solution, small peptides are thought to exist in a multiplicity of transient conformational states in dynamic equilibrium (6, 7). Antibodies raised against short peptides often react well with the native protein at the region of sequence homology, when this region is located at the surface of the molecule (8-10). It may also be necessary to restrict the conformational freedom of immunizing peptides in order to obtain antibodies of the same specificity as those induced by the proteins themselves.To compare the specificity of the anti-protein response with the anti-peptide response, peptides were synthesized corresponding to an equivalent epitope of three foot-andmouth disease virus (FMDV) strains, each belonging to a different serotype of the virus. It was assumed that anti-virus sera raised against different viral serotypes would not crossreact at a neutralization epitope. The diversity of antibodies directed to a single epitope was compared for each pair of antisera (anti-peptide and anti-virus). The specificity of each of the anti-peptide sera was inferred from the relative reactivity for the homologous compared to the heterologous peptide.A replacement set was synthesized consisting of all of the peptides derived by ...

A set of monoclonal antibodies was used to isolate nonneutralizable foot-and-mouth disease virus variants, and the RNAs of the variants were sequenced. Cross-neutralization studies and mapping of the amino acid changes indicated two major antigenic sites. The first site was trypsin sensitive and included the VP1 140 to 160 sequence. The second site was trypsin insensitive and included mainly VP3 residues. Two minor sites were located near VP1 169 and on the C terminus of VP1. Comparison with poliovirus type 1 and human rhinovirus 14 showed a similarity in the immunogenicity of comparable sites on the viruses.

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