General method for the rapid solid-phase synthesis of large numbers of peptides: specificity of antigen-antibody interaction at the level of individual amino acids.

Abstract

A novel yet simple method is described that facilitates the synthesis of large numbers of peptides to the extent that the synthesis process need no longer be the limiting factor in many studies involving peptides. By using the methods described, 10-20 mg of 248 different 13-residue peptides representing single amino acid variants of a segment of the hemagglutinin protein (HAI) have been prepared and characterized in less than 4 weeks. Through examination of the binding of these analogs to monoclonal antibodies raised against residues 75-110 of HAl, it was found that a single amino acid, aspartic acid at position 101, is of unique importance to the interaction. Two other residues, aspartic acid-104 and alanine-106, were found to play a lesser but significant role in the binding interaction. Other single positional residue variations appear to be of little or no importance.Over the past 5 years there has been an escalating need for synthetic peptides in a wide variety of applications, including the development of synthetic peptide vaccines (1-5), the detailed study of antigen-antibody interactions (6-11), the preparation of optimal analogs of biologically active peptides (12)(13)(14), the optimization of peptide antigens of clinical diagnostic utility (15-17), the mapping of protein products of brain-specific genes (18,19), and the study of protein conformational parameters (20). In the majority of these studies, the limiting factor has been the availability and cost of the desired peptides. Clearly, these studies would be greatly facilitated if synthetic methods were available that would permit the synthesis of larger numbers of peptides in a more cost efficient manner and in a shorter period of time per peptide. While it is currently possible to produce large numbers ofpeptides (i.e., hundreds) using available methods, the effort is so time-consuming and expensive that to actually carry out this number of syntheses is impractical, even in especially well-equipped laboratories. The first part of this paper describes a general method that can be utilized for the rapid (2-6 weeks) synthesis of large numbers of peptides (>100) in quantities that satisfy most needs (>10 mg).It is known from recent studies that antibodies raised against synthetic peptides frequently react with sites in the parent protein (1,2,11). These findings allow more detailed understanding of immunological interactions. Thus, instead of defining the reactivity of a protein antigen at the whole protein or subunit level, antigen-antibody interactions can be localized within the antigen to a short sequence of immunological importance (7,11). The next step is the refinement of our understanding of the antigenic sites of proteins or peptides at the level of individual amino acids. To illustrate the utility of the synthesis procedures presented here, the second part of this paper presents a detailed study of peptide antigen-antibody interactions. In the example presented, 248 individual peptides were synthesized and used in a study of one part...

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