Characterization of antigen-antibody interactions using single substitution analogs and mixture-based synthetic combinatorial libraries

J. R. Appel, G. D. Campbell, J. Buencamino, R. A. Houghten, Clemencia Pinilla

Research output: Contribution to journalArticlepeer-review

7 Scopus citations

Abstract

In an effort to use monoclonal antibodies (mAbs) as selective probes for early detection of breast cancer, the specificities of a number of antipeptide mAbs have been studied at the individual amino acid level using single substitution peptide analogs and peptide combinatorial libraries. In this study, the mapping results are presented for mAb172-12A4, which was raised against the haptenic peptide LGSGAFGTIYKG(C), corresponding to residues 138-149 of the oncogene v-erbB. This peptide is homologous with a region in epidermal growth factor receptor (EGFR) and human oncogene c-erbB- 2, and contains the ATP binding motif that is common among protein kinases. The substitution profile of this interaction correlated well with the results from the screening of hexa- and decapeptide positional scanning libraries. Based on the results of this mAb's specificity for the antigenic determinant (-AFGTIYK-), proteins that have sequence homology were found from a database search of human sequences. Thirty-two unique peptide sequences, a majority of which was from protein kinases, were synthesized and tested for recognition by mAb 172-12A4. Eleven peptides had activities that differed from the original peptide by less than an order of magnitude, and the activities for 29 of the 32 (90%) could be accurately predicted based on the individual substitution analog results. While both epitope mapping approaches address the amino acid level of mAb specificity, positional scanning libraries offer an advantage of identifying the positional importance of each antigenic determinant residue without any prior knowledge of the mAb's specificity. The fine specificity mapping of peptide-specific mAbs using the synthetic tools illustrated here will be useful for the development of immunodiagnostics that detect cancer-related proteins in clinical samples.

Original languageEnglish (US)
Pages (from-to)346-355
Number of pages10
JournalJournal of Peptide Research
Volume52
Issue number5
DOIs
StatePublished - 1998
Externally publishedYes

Keywords

  • Antigen-antibody interactions
  • Cancer detection
  • Oncogenes
  • Positional scanning
  • Synthetic combinatorial libraries

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