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

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.

• Authors: Euan Murray, Ekaterina O McKenna, Lindsay R Burch, John Dillon, Pat Langridge-Smith, Walter Kolch, Andrew Pitt, Ted R Hupp
• Journal: Biochemistry (Biochemistry) Vol. 46 Issue 48 Pg. 13742-51 (Dec 04 2007) ISSN: 0006-2960 [Print] United States
• PMID: 17994709 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• AGR2 protein, human
• Aptamers, Peptide
• Biomarkers
• Mucoproteins
• Oncogene Proteins
• Proteins

Topics

• Amino Acid Sequence
• Animals
• Aptamers, Peptide (chemistry)
• Biomarkers (chemistry)
• Chromatography, Affinity
• Combinatorial Chemistry Techniques
• Enzyme-Linked Immunosorbent Assay
• Humans
• Molecular Sequence Data
• Mucoproteins
• Oncogene Proteins
• Protein Array Analysis
• Proteins (chemistry, isolation & purification)
• Swine