Chemically unambiguous peptide immunogen: preparation, orientation and antigenicity of purified peptide conjugated to the multiple antigen peptide system.
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 alpha (TGF alpha) 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 TGF alpha 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.
Lu
Y A
YA
Rockefeller University, New York, NY 10021.
Clavijo
P
P
Galantino
M
M
Shen
Z Y
ZY
Liu
W
W
Tam
J P
JP
D000595:Amino Acid Sequence; D000818:Animals; D000918:Antibody Specificity / Q000235:genetics; D004797:Enzyme-Linked Immunosorbent Assay; D008969:Molecular Sequence Data; D009154:Mutation; D010455:Peptides; D011817:Rabbits; D016211:Transforming Growth Factor alpha / Q000276:immunology; D014614:Vaccines, Synthetic / Q000138:chemical synthesis* / Q000276:immunology
D010455:Peptides; D016211:Transforming Growth Factor alpha; D014614:Vaccines, Synthetic