Microarray-formatted clinical biomarker assay development using peptide aptamers to anterior gradient-2

Euan Murray, Ekaterina O. McKenna, Lindsay R. Burch, John Dillon, Pat Langridge-Smith, Walter Kolch, Andrew Pitt, Ted R. Hupp

Research output: Contribution to journalArticle

Abstract

Anterior gradient-2 protein was identified using proteomic technologies as a p53 inhibitor which is overexpressed in human cancers, and this protein presents a novel pro-oncogenic target with which to develop diagnostic assays for biomarker detection in clinical tissue. Combinatorial phage-peptide libraries were used to select 12 amino acid polypeptide aptamers toward anterior gradient-2 to determine whether methods can be developed to affinity purify the protein from clinical biopsies. Selecting phage aptamers through four rounds of screening on recombinant human anterior gradient-2 protein identified two classes of peptide ligand that bind to distinct epitopes on anterior gradient-2 protein in an immunoblot. Synthetic biotinylated peptide aptamers bound in an ELISA format to anterior gradient-2, and substitution mutagenesis further minimized one polypeptide aptamer to a hexapeptide core. Aptamers containing this latter consensus sequence could be used to affinity purify to homogeneity human anterior gradient-2 protein from a single clinical biopsy. The spotting of a panel of peptide aptamers onto a protein microarray matrix could be used to quantify anterior gradient-2 protein from crude clinical biopsy lysates, providing a format for quantitative screening. These data highlight the utility of peptide combinatorial libraries to acquire rapidly a high-affinity ligand that can selectively bind a target protein from a clinical biopsy and provide a technological approach for clinical biomarker assay development in an aptamer microarray format.
Original languageEnglish
Pages (from-to)13742-13751
Number of pages10
JournalBiochemistry
Volume46
Issue number48
Early online date10 Nov 2007
DOIs
Publication statusPublished - 4 Dec 2007

Keywords

  • amino acid sequence
  • animals
  • peptide aptamers
  • biological markers
  • affinity chromatography
  • combinatorial chemistry techniques
  • enzyme-linked immunosorbent assay
  • humans
  • molecular sequence data
  • protein array analysis
  • proteins
  • swine

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