Comparative Analysis of Multiplex Platforms for Detecting Vitreous Biomarkers in Diabetic Retinopathy

Abstract

To evaluate the feasibility of using the Proximity Extension Assay (PEA) platform to detect biomarkers in vitreous and to compare the findings with results obtained with an electrochemiluminescent (ECL) sandwich immunoassay. Methods: Vitreous samples from patients with proliferative diabetic retinopathy (PDR) and non-diabetic controls were tested using two different proteomics platforms. Fortyone assays were completed with the ECL platform and 459 with the PEA platform. Spearman's rank correlation coefficient (r s) was used to determine the direction and strength of the relationship between protein levels detected by both platforms. Results: Three hundred sixty-six PEA assays detected the tested protein in at least 25% of samples, and the difference in protein abundance between PDR and controls was statistically significant for 262 assays. Seventeen ECL assays yielded a detection rate ≥ 25%, and the difference in protein concentration between PDR and controls was statistically significant for 13 proteins. There was a subset of proteins that were detected by both platforms, and for those the Spearman's correlation coefficient was higher than 0.8. Conclusions: PEA is suitable for the analysis of vitreous samples, showing a strong correlation with the ECL platform. The detection rate of PEA panels was higher than the panels tested with ECL. The levels of several proinflammatory and angiogenic cytokines were significantly higher in PDR vitreous compared to controls. Translational Relevance: This study provides new information on the yields of smallvolume assays that can detect proteins of interest in ocular specimens, and it identifies patterns of cytokine dysregulation in PDR.

The Vitreous Ecosystem in Diabetic Retinopathy: Insight into the Patho-Mechanisms of Disease

“…For targeted analysis, enzyme-linked immunosorbent assay (ELISA) by far has been the most common way to detect and analyze vitreous cytokines, having replaced radioimmunoassay [16], but in the early 2000s, multiplex methods arrived which were more cost-effective as well as efficient in measuring multiple mediators simultaneously [17][18][19]. Methods using multiplex technology that enhance reproducibility and detect proteins in lower concentrations such as electrochemiluminescent (ECL) sandwich immunoassay and proximity extension assay (PEA) are now widely available [20]. These techniques allow many proteins to be analyzed simultaneously with as small as 1 µL of volume [20].…”

“…Methods using multiplex technology that enhance reproducibility and detect proteins in lower concentrations such as electrochemiluminescent (ECL) sandwich immunoassay and proximity extension assay (PEA) are now widely available [20]. These techniques allow many proteins to be analyzed simultaneously with as small as 1 µL of volume [20].…”

Vitreous Biomarkers for Proliferative Vitreoretinopathy Prognostication in Patients Undergoing Primary Retinal Detachment Repair

“…A full list of potential protein biomarkers included in these panels and associated assay validation data (e.g., limit of detection [LOD], lower and upper limits of quantification, within- and between-run precision coefficient of variation) can be found on the manufacturer's website ( https://www.olink.com ) and Supplementary Tables S1 and S2 . 5 , 6 Each panel simultaneously analyzes 92 proteins utilizing 1 µL of biological sample and reports Normalized Protein eXpression (NPX) values 7 (Olink Proteomics, Uppsala, Sweden). The NPX is an arbitrary unit on a log base 2 scale wherein higher NPX values correlate with higher protein concentrations, where each point difference is equivalent to a twofold change in protein concentration.…”

Advances in aqueous humor proteomics for biomarker discovery and disease mechanisms exploration: a spotlight on primary open angle glaucoma

“…Novel affinity proteomics discovery platforms, such as SomaScan 11k and Olink Explore HT, enable the analysis of thousands of protein targets in a semi-quantitative manner—without any sample pre-processing. While being largely developed for the analysis of blood-derived samples, the platforms offer access to sample matrices beyond those, including saliva ( Scholtz et al, 2020 ), urine ( Daza et al, 2023 ), ascites ( Finkernagel et al, 2019 ) and many other bodily fluids including AH and vitreous humor (VH) ( Lamy et al, 2020 ; Peng et al, 2023 ; Wolf et al, 2023 ).…”