Development and Evaluation of an Ultrasensitive Free VEGF-A Immunoassay for Analysis of Human Aqueous Humor

Abstract

Aim: Novel bifunctional VEGF-A neutralizing therapies are being developed for the treatment of retinal vascular diseases such as age-related macular degeneration and diabetic retinopathy. In developing new therapeutic drugs, only small aqueous humor sample volumes are available for analyzing several parameters. Highly sensitive detection methods must be applied in analyzing VEGF-A levels in ocular fluids in order to demonstrate VEGF-A suppression following drug administration. Experimental: A highly sensitive immunoassay for VEGF-A was developed on the single molecule array (Simoa) platform, and validated before being used for the analysis of clinical aqueous humor samples from patients treated with anti-VEGF-A therapeutics. Results: This highly sensitive immunoassay allows the detection of baseline VEGF-A levels and suppression effects after drug administration, even in sample volumes as low as 12 μl. Conclusion: The Simoa VEGF-A assay is a valuable tool for the reliable monitoring of VEGF-A suppression after intravitreal administration of anti-VEGF-A drugs.

Digital detection of proteins

“…For example, Göpfert and co-workers developed a DBA assay for measuring VEGF-A in aqueous humor from the eyes of human subjects. 151 They used the greater sensitivity of this digital assay to measure reductions in the concentration of VEGF in the ocular fluid caused by administration of a therapeutic antibody that targets this growth factor; conventional bead immunoassays were not able to detect these reductions. The sensitivity of homebrew DBA has also been used to measure proteins in other challenging samples where protein concentrations and sample volumes may be low, such as breath condensate, 152 sweat, 153 saliva, 154 and stool.…”