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 ...

Structure-based design of synthetic peptide-based molecules that mimic the functional site of natural proteins, plays an important role in drug discovery nowadays. [1][2][3][4][5] Their application is widespread, ranging from synthetic antiviral, [3, 6] antifertility, [1,2,7] or antitumor [2,7, 8] agents to therapeutic agents that are able to mimic [9] or disrupt [4, 10] protein-protein interactions. A variety of structural mimics exist for a-helices, [11,12] b-turns or hairpins, [11, 13] and b-sheets. [11,14] However, more complex topologies, like four-helix bundles, [15] are often needed in order to mimic protein function adequately.[16] The total synthesis of such complex structures is generally demanding; this limits their application and emphasizes the need for high-efficiency synthetic strategies. In this communication, we describe a onestep procedure for the immobilization of (multiple) peptide loops onto a synthetic scaffold (Scheme 1) starting from a linear peptide. The reaction is extremely fast and clean and runs very well with linear peptides that are 2-30 amino acids long (> 30 not tested). It is compatible with all possible unprotected side-chain functionalities (except for free cysteine). It therefore avoids the need for complex synthetic strategies and this makes the reaction highly versatile with a very wide scope.As part of our research program on the mapping and reconstruction of the discontinuous epitope of follicle-stimulating hormone (FSH), [17] which is a heterodimeric member of the cysteine-knot protein family, [18] we recently discovered the fast and quantitative cyclization of dicysteine-containing peptides upon their treatment with a,a'-dibromoxylenes (T2). In organic solvents such as ACN, the reaction is rather slow and unselective, [19] but it becomes unusually fast and entirely selective for cysteines when performed in aqueous solutions.[20] For example, treatment of a 0.5 mm solution of the peptide *CRVPGDAHHADSLC# (1 a, where * = acetyl and # = amide) with 1.05 equiv of m-T2 in a 1:7 mixture of ACN/NH 4 HCO 3 (20 mm, pH 7.8) gives the corresponding monocyclic product 2 a with > 80 % yield in less than 15 min at RT (see Table 1). The corresponding intramolecular SS-dimer 3 is not formed (< 5 %) as oxidative cyclization is not competitive under these conditions. There is no doubt that the reaction takes place exclusively at the free sulfhydryl groups, since corresponding peptides without sulfhydryl groups [21] do not react at all with T2 scaffolds in the solvent system used.The difference in reactivity amongst various dicysteine-containing peptides that we have studied is negligible. The half-lives of peptides 1 a-g in the reaction with m-T2 vary only slightly (t 1/2 = 1.4-3.0 min, see Table 1), despite the fact that their length (14-42) and the number of amino acids that separate the two cysteines (0-22) are very different. In sharp contrast to this, there is a large difference in reactivity amongst different scaffolds. o-T2 (average t 1/2 = 1.4 min) is slightly more reactiv...

Chimpanzees are susceptible to infection by di- No antibodies to the carboxyl terminus of HTLV-IUB gpl20 were observed in sera of chimpanzees inoculated with HTLV-ITIRF or with the brain-tissue strain, and those sera did not neutralize HTLV-IIIB. A rabbit immunized with the C-terminal portion of gpl20 acquired neutralizing antibodies that bound to four domains of the HTLV-UIB external envelope as analyzed by reactivity to 536 overlapping nonapeptides of gpl20. One of these domains in the variable region V3, with the amino acid sequence IRIQRGPGRAFVTIG (amino acids 307-321), bound to all chimpanzee sera that neutralized HTLV-IIIB but not to the serum of the HTLV-IUIRF-inoculated chimpanzee that did not neutralize HTLV-UIB. The HTLV-IIIRF sequence at the same location, ITKGPGRVIYA, was recognized by the serum of the HTLV-UIERF-inoculated chimpanzee but not by any sera of the HTLV-IUB-inoculated or LAV-i-inoculated chimpanzees. The HTLV-UIB residues RIQR and AFV and the HTLV-IIRF residues lysine and VIYA, flanking a highly conserved (3-turn (GPGR), appear to be critical for antibody binding and subsequent type-specific virus neutralization. This neutralization epitope, putatively consisting of a loop between two cysteine residues (amino acids 296 and 331) connected by a disulfide bond, is immunodominant in HIV-1-infected chimpanzees and induces antibodies restricted to the homologous viral strain.

Abstract.A converted form of the normal cellular prion protein (PrP) accumulates in the brains of sheep with scrapie. We describe an immunohistochemical method for identifying scrapie-associated PrP (PrPsc) in periodate-lysine-paraformaldehyde-fixed brain tissue, which provides adequate preservation of tissue morphology. After pretreatment of tissue sections with formic acid and hydrated autoclaving, we located PrPk in the brains of 50 sheep with natural scrapie by use of antipeptide antisera raised against ovine PrP. No PrP was seen in 20 sheep without histopathologic signs of scrapie. PrPSc that did not stain for amyloid was present in the cytoplasm and at the cell membrane of both neurons and astrocytes. Large amounts of PrPSc were seen at the cell membrane of neurons in the medulla oblongata and pons, whereas PrP" accumulated at the cell membrane of astrocytes of the glial limitans in all brain regions. PrP" that stained for amyloid was located in the walls of blood vessels and perivascularly in the brains of 32 (64%) of 50 sheep, mainly in the thalamus and never in the pons or medulla oblongata. No apparent topographic relationship existed between PrPSc that stained for amyloid and PrPSc accumulation associated with neurons or astrocytes. In all scrapie-affected sheep, PrPSc was present in brain regions with vacuolation, but it could also be detected in regions with minimal or no vacuolation. We conclude that the immunohistochemical detection of PrP can be an important confirmative test in scrapie diagnosis.