Optimization of immunoassay parameters in multiplex in the high throughput protein detection technique Proximity Extension Assay

Abstract: The ability to detect protein-based biomarkers, which are linked to different diseases like colorectal cancer, is very important as a diagnostic tool. Usually complex biological samples like blood are studied which will contribute to different technical issues when performing an assay. The aim with the project is to optimize and develop the high throughput protein detection technique Proximity Extension Assay, PEA, into a 96-plex panel, in hopes of discovering an expression profile for colorectal cancer. PEA was developed by Olink Bioscience and allows specific proteins in a sample to be quantitatively transformed into nucleic acid sequences that are subsequently detected and quantified with real-time PCR. Two proximity probes containing oligonucleotide sequences bind pairwise to target protein and when brought in proximity, a DNA polymerase will extend a hybridization arm from one probe over to the second forming a double-stranded DNA sequence that can serve as a template in real-time PCR. The results showed that there is no significant difference in sensitivity, specificity, recovery or efficiency between assays performed in single plex, lower or higher degree of multiplex. Higher sensitivity of the assay was achieved by optimization of factors such as, concentrations of proximity probes and hybridization oligo arm. The results also show that the recovery will not be affected by higher concentration of plasma or by using other assay formats. Work proceeds to develop a 96-plex panel with just as high sensitivity and recovery, which would make PEA the most, multiplexed immunoassay with high sensitivity so far.