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Extracellular Protein Microarray Technology for High Throughput Detection of Low Affinity Receptor-Ligand Interactions
Published on: January 7, 2019
Microarray-formatted clinical biomarker assay development using peptide aptamers to anterior gradient-2.
Euan Murray1, Ekaterina O McKenna, Lindsay R Burch
1CRUK p53 Signal Transduction Group, Cell Signalling Unit, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh EH4 2XR, United Kingdom.
Biochemistry
|November 13, 2007
Summary
Researchers developed peptide aptamers to detect anterior gradient-2 protein, a cancer biomarker. These aptamers can purify and quantify the protein from clinical samples for diagnostic assays.
Area of Science:
- Biochemistry
- Proteomics
- Molecular Biology
Background:
- Anterior gradient-2 (AG-2) protein is a p53 inhibitor overexpressed in human cancers.
- AG-2 presents a potential pro-oncogenic target for cancer diagnostics.
- Developing methods to detect AG-2 in clinical tissues is crucial.
Purpose of the Study:
- To develop diagnostic assays for biomarker detection of anterior gradient-2 protein in clinical tissue.
- To determine if peptide aptamers can affinity purify AG-2 from clinical biopsies.
- To establish a technological approach for quantitative screening of AG-2.
Main Methods:
- Utilized combinatorial phage-peptide libraries to select aptamers against AG-2.
- Employed screening on recombinant human AG-2 protein and immunoblotting.
- Used ELISA and substitution mutagenesis to refine aptamer binding and minimize sequence.
- Developed aptamer-based protein microarrays for quantification.
Main Results:
- Identified two classes of peptide ligands binding distinct epitopes on AG-2.
- Synthetic biotinylated aptamers showed binding to AG-2 in ELISA.
- Minimized one aptamer to a hexapeptide core with high affinity.
- Demonstrated purification of AG-2 to homogeneity from clinical biopsies using aptamers.
- Showcased aptamer microarrays for quantifying AG-2 in clinical lysates.
Conclusions:
- Peptide aptamers can be rapidly acquired to selectively bind target proteins like AG-2.
- Developed a method for affinity purification of AG-2 from clinical biopsies.
- Established a technological approach for developing quantitative aptamer microarray-based biomarker assays.
