Validation of a Novel Cytokine Multiplex Assay for Vitreous Analysis in Diabetic Retinopathy

Abstract

Purpose : Vascular endothelial growth factor (VEGF) and also cytokines and chemokines have been identified as key players in disease development and progression of diabetic retinopathy (DR) and other retinal angiogenic diseases. As such, these factors may not only represent therapeutic targets but also biomarkers for patient stratification or treatment monitoring.
However, the analysis of biomarkers from ocular fluids in the clinical setting is limited by low sample quantities. This study describes the analytical validation of a novel bead based microarray for the simultaneous quantification of IL-1β, IL-2, IL-6, IL-8, TNFα, VEGF-A from only minute amounts of ocular fluids. Furthermore, we show the analysis of vitreous humour samples from a small cohort of Diabetes mellitus (DM) patients with and without DR.

Methods : The AYOXXA Lunaris^TM System enables reliable multiplex analysis from minute amounts of biological samples. Capture antibody coated microspheres are immobilized in micro-sized cavities of a biochip. The assay follows the sandwich ELISA principle. By using fluorescence-labeled detection reagents signal intensity of every single reaction per microsphere is recorded using the AYOXXA reader and concentrations calculated with the Lunaris™ Analysis Software. For the analysis, 5 µl of prediluted vitreous humour samples were subjected to the standard workflow with the Lunaris™ Human 6-plex Ophthalmology Kit.

Results : The novel multiplex assay demonstrated high sensitivity, precision and accuracy within and between assay runs. In vitreous humour samples elevated levels of VEGF-A, IL-6 and IL-8 were observed in DR patients when compared to non-diabetic controls.

Conclusions : This study demonstrates the analytical reliability of the Lunaris™ platform for the evaluation of biomarkers of DR such as VEGF in small amounts of ocular fluids. This technology will enable biomarker analysis close to the origin of the disease and can become a feasible patient monitoring or stratification tool to evaluate treatment and disease progression.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.