title: 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.affiliation: CRUK p53 Signal Transduction Group, Cell Signalling Unit, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh EH4 2XR, United Kingdom.
authors.forename: Euan
authors.initials: E
authors.lastname: Murray
authors.forename: Ekaterina O
authors.initials: EO
authors.lastname: McKenna
authors.forename: Lindsay R
authors.initials: LR
authors.lastname: Burch
authors.forename: John
authors.initials: J
authors.lastname: Dillon
authors.forename: Pat
authors.initials: P
authors.lastname: Langridge-Smith
authors.forename: Walter
authors.initials: W
authors.lastname: Kolch
authors.forename: Andrew
authors.initials: A
authors.lastname: Pitt
authors.forename: Ted R
authors.initials: TR
authors.lastname: Hupp
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D052158:Aptamers, Peptide / Q000737:chemistry*; D015415:Biomarkers / Q000737:chemistry*; D002846:Chromatography, Affinity; D020650:Combinatorial Chemistry Techniques; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D008969:Molecular Sequence Data; D009088:Mucoproteins; D015513:Oncogene Proteins; D040081:Protein Array Analysis; D011506:Proteins / Q000737:chemistry* / Q000302:isolation & purification; D013552:Swine
chemical_list: C502296:AGR2 protein, human; D052158:Aptamers, Peptide; D015415:Biomarkers; D009088:Mucoproteins; D015513:Oncogene Proteins; D011506:Proteins