Microarray-formatted clinical biomarker assay development using peptide aptamers to anterior gradient-2.
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.
Murray
Euan
E
CRUK p53 Signal Transduction Group, Cell Signalling Unit, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh EH4 2XR, United Kingdom.
McKenna
Ekaterina O
EO
Burch
Lindsay R
LR
Dillon
John
J
Langridge-Smith
Pat
P
Kolch
Walter
W
Pitt
Andrew
A
Hupp
Ted R
TR
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
C502296:AGR2 protein, human; D052158:Aptamers, Peptide; D015415:Biomarkers; D009088:Mucoproteins; D015513:Oncogene Proteins; D011506:Proteins