H. Mario Geysen

Strategies for epitope analysis using peptide synthesis

A recently developed approach to the synthesis and ELISA screening of large numbers of peptides is described. The method has created the opportunity to tackle questions about the sites and specificity of antigenic determinants which were formerly thought to be too difficult to answer. The various strategies for application of this method are described along with examples of their successful use. They include a procedure for locating all the continuous antigenic peptides of a protein antigen, and the identification of non-replaceable amino acid residues within an antigenic peptide. An approach to the determination of amino acid residues involved in the epitope for any monoclonal antibody is also described. These strategies open up the prospect of rapid mapping of the antigenic properties of hitherto poorly understood antigens.

A priori delineation of a peptide which mimics a discontinuous antigenic determinant

A technique was developed for identifying peptides with high affinity for a given antibody. By testing a monoclonal antibody directed against a discontinuous antigenic determinant on foot-and-mouth disease virus, peptides mimicking the determinant were identified even though the tertiary structure of the proteins comprising the virus capsid is unknown. The allowable variations in spacing and stereochemistry of the peptides shown to mimic this epitope suggest protein folding in which amino acid residues from three regions, distant from one another in the primary sequence, are brought into close proximity at this epitope. The technique has potential for identification of peptides which will bind with high affinity to receptors other than antibody molecules.

Multi-pin peptide synthesis strategy for T cell determinant analysis

Techniques to synthesize many peptides simultaneously exist, however their individual cleavage and subsequent purification constitutes a bottleneck to total throughput. Biological screening of peptides is generally carried out at physiological pH in aqueous solutions. However, peptides, unless individually purified are usually contaminated by residual compounds used in their preparation such as trifluoroacetic acid, organic solvents, scavengers etc. In testing with cellular systems, such as T cell determinant analysis, such contaminations must be rigorously excluded. We have extended the pin synthesis technique of synthesizing and screening large number of peptides (Geysen et al., 1984) to the analysis of T cell determinants. Peptides can be synthesized on polyethylene pins, the side chain protective groups removed and the peptides washed free of contaminants. A linker system stable under these conditions can then be triggered to cleave the peptides from the pins in an aqueous solution at neutral pH. This strategy enables the rapid mapping of T cell determinants. It is also applicable to other systems where large numbers of solution phase peptides are required, for example, in the study of hormone analogues.

The Chemistry and Mechanism of Antibody Binding to Protein Antigens

Publisher Summary There are already a number of authoritative reviews on protein immunogenicity. The chapter discuses the chemical nature of the recognition process associated with the production of antibodies. Detailed crystallographic information on the structure of antibody–antigen complexes is described in the chapter. Although there are many different ways to identify protein epitopes, the chapter focuses primarily on results from X-ray crystallography and peptide mapping, because these currently provide the most suitable data base for the consideration of the chemistry and mechanism of antibody binding to protein antigens. Most antibodies raised against intact proteins recognize conformational sites, consequently, do not bind peptides or proteolytic fragments with high affinity. Yet, some antibodies raised against native protein antigens do bind to peptide homologs of the protein sequence, allowing the chemistry of these sites to be probed at the resolution of single amino acid side chains. Some antibodies raised against the native protein appear to recognize the denatured forms of the protein only. Crystallographic approaches to characterize antibody–antigen complexes are limited by practical constraints, resulting from the experimental difficulties of protein crystallization and from the extensive time and equipment requirements. Nevertheless, the crystallographic structures of antibody complexes with protein antigens provide a wealth of information with which all other biochemical and immunological experiments must be reconciled.

Screening chemically synthesized peptide libraries for biologically-relevant molecules

Abstract Practical strategies for the synthesis and testing of large peptide libraries are described. The relationship between the number of monomers (amino acids) used to generate diversity and the magnitude of the synthesis and testing required is evaluated. Peptide optimization methods using strategies of replacement by non-natural and D-optical isomers are demonstrated.

The antibody response to myoglobin—I. Systematic synthesis of myoglobin peptides reveals location and substructure of species-dependent continuous antigenic determinants

Sets of peptides representing all the possible hepta-, octa-, nona- and decapeptides of sperm whale myoglobin were synthesized. An ELISA method was used to detect the ability of antibodies, present in antisera raised against native sperm whale myoglobin, to bind to these peptides. Antisera made in two species were compared. It was found that the peptides recognized by the antibodies were a function of the species in which the antiserum was prepared and of the individual outbred member of that species. Peptides corresponding to surface epitopes of the native antigen were identified by reacting the antisera with native antigen prior to ELISA testing on peptides. More detailed analysis of one epitope revealed that, for some sera, a leucine residue which is facing inwards in the crystal structure is critical for the binding of antibody to the peptide. This suggests that binding between native antigen and antibody can require a restructuring of the native antigen.

Grafted supports used with the multipin method of peptide synthesis

Abstract Radiation grafting of a range of monomers allows a diversity of surface characteristics to be generated on a common plastic support. Taken together with the modular 8-column, 12-row format used with the multipin method of peptide synthesis, many thousands of peptides can be generated not only for epitope mapping and other solid-phase applications but also to produce larger quantities of cleaved peptides.

Monoclonal antipeptide antibodies: Affinity and kinetic rate constants measured for the peptide and the cognate protein using a biosensor technology

The interaction of antipeptide antibodies with the corresponding peptide and the cognate protein has been compared using a novel biosensor technology (BIAcore, Pharmacia). The peptide corresponds to residues 110-135 of the coat protein of tobacco mosaic virus, known to encompass an alpha-helical region reactive with antiprotein antibodies. A panel of 33 monoclonal antibodies raised against the peptide was studied and the epitope recognized by these antibodies was determined by the pepscan method. Further discrimination between the antibodies was performed by measurements of association and dissociation kinetic constants. Several antibodies showed an heterogeneous binding profile when reacting with the 25 residue long peptide but not with a shorter 10 residue peptide suggesting that they recognized different conformational states in the longer peptide. Equilibrium affinity constants were calculated for five of the antibodies and were found to be 10-50 times higher for the peptide than for the protein, the difference being caused mainly by a lower association rate constant.

Scanning for T helper epitopes with human PBMC using pools of short synthetic peptides

Major T helper epitopes of medically important antigens can be located by measuring the proliferative responses of human peripheral blood mononuclear cells (PBMC) to pools of short synthetic peptides. The length and endings of the peptides used were shown to be critical for success in identifying Th cell epitopes. Many epitopes would be missed if either long (31mers) or short (less than 12mers) peptides were used. Pools of 14 and 16mers were more efficient than 12mers spanning the same region, however, for a promiscuous Th cell epitope of tetanus toxin (tt 947-967), two of three donors tested did not respond to 18mers or shorter peptides spanning this region. Although peptides with either unblocked or blocked ends were stimulatory, peptides with blocked ends were generally more efficient. The peptide concentration and number of available APC were also found affect the efficiency of the proliferation assay as a measure of peptide recognition by Th cells. Two screenings of the entire set of tetanus toxin peptide pools using different samples of PBMC from the same donor identified common major stimulatory regions. Thus, PBMC and peptide pools can be used for the reproducible identification of Th cell epitopes. After immunization with tetanus toxoid (TT), peptide-responsive cells increased in frequency in parallel to the increase in TT responsive cells, indicating that the peptide-responsive cells were primed by TT.

Endothelin receptor ligands. replacement net approach to SAR determination of potent hexapeptides

Abstract We determined the SAR of the potent hexapeptide endothelin ligand Ac-Dphe-Orn-Asp-Ile-Ile-Trp-OH1 (1) through the systematic replacement of each residue with 50 amino acid substitutes. Multipin peptide synthesis methods allowed us to rapidly synthesize and then screen all 300 analogues.