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Chemically unambiguous peptide immunogen: Preparation, orientation and antigenicity of purified peptide conjugated to the multiple antigen peptide system

1991; Elsevier BV; Volume: 28; Issue: 6 Linguagem: Inglês

10.1016/0161-5890(91)90131-3

1872-9142

Lu Yi-An, Pedro Clavijo, Mauro Galantino, Shen Zhi-yi, Wen Liu, James P. Tam,

Glycosylation and Glycoproteins Research

We described a novel and simple approach to prepare chemically unambiguous peptide immunogen using the multiple antigen peptide (MAP) approach. This approach requires the conjugation of two purified components: a chloroacetylated oligomeric lysine core matrix and a synthetic peptide containing cysteine at either the carboxyl or amino terminus. The resulting MAP is structurally unambiguous and contains a quantifiable amount of peptide antigens. Furthermore, this method also provides a flexible strategy to link a peptide antigen to the core matrix at the desirable orientation to mimic the native molecule. The carboxyl fragment 43–50 of human transforming growth factor α (TGFa) was used as a test model for this approach. Antipeptide antibodies did not recognize the "reverse immunogen" in which the peptide was attached to the MAP core matrix at a reverse orientation. To determine the specificity of the antibodies, we used two series of point-substituted TGFα analogs containing either alanine or the corresponding d-amino acid replacement to map the antigenic site. The alanine analogs were used to determine the contribution of the side chain while the d-amino acid analogs were used to determine the importance of backbone conformation. The antigen site was found to consist of four residues (Asp47-Leu48-Leu49-Ala50) at the distal end of the peptide-MAP conjugate. The results provide a clear explanation for the specificity of the antipeptide antibodies and their failure to recognize the "reverse immunogen" since the distal and the flexible end of the peptide-MAP construct constitutes the antigenic site. Furthermore, our results also suggests a strategy of placing the antigenic portion of a short-peptide at the distal end in the MAP approach to prepare immunogen.