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Tobias Wimmer, Constanze Stumpf, Eva Schroeter, Birgit Lorenz, Knut Stieger; Development of a BRET-based technique for VEGF quantification. Invest. Ophthalmol. Vis. Sci. 2016;57(12):5052.
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© ARVO (1962-2015); The Authors (2016-present)
The major stimulus of neovascular disorders like the age related macular degeneration (AMD), the diabetic retinopathie (DR) or retinopathie of prematurity (ROP) is the hypoxia induced upregulation of vascular endothelial growth factor (VEGF) expression which leads to an uncontrolled formation of new, immature blood vessels in the eye. The increased VEGF level can be neutralized with anti-VEGF molecules like Avastin®, Lucentis® or Eyelea®. Morphologic changes measured with ocular coherence tomography and functional alterations as measured with visual acuity are occurring delayed, after the VEGF concentration has increased again. The aim of the study is to develop a method to continuously quantify VEGF introcularly.
An anti-VEGF single chain variable fragment (scFv) was used as parental molecule to generate a BRET-based, VEGF binding Biosensor, by fusing a Renilla Luciferase (RLuc8) to the N-terminal part of the anti-VEGF scFv as the BRET donor and fluorophors (YFP:BRET1, GFP2:BRET2) to the C-terminal part of the scFv as the BRET acceptor. HEK293 were transfected with these biosensor constructs and expression was verified by luciferase activity assay and fluorescence microscopy. The VEGF binding and the concentration dependent change in BRET ratio was measured with a plate reader in dual luminescence mode using the filter sets for BRET1&2.
The BRET ratio measured for the BRET1 construct was 0.440 ± 0.020 and 0.785 ± 0.039 for the BRET2 construct. The VEGF binding capacity per 1x106 RLU (relative light units) was 20.15 pg ± 2.20 pg for the BRET1 variant and 20.29 pg ± 6.60 pg for the BRET2 biosensor. The linear range of the VEGF dependent BRET ratio change was evaluated from 500 fg/ml to 50 pg/ml for the BRET1 molecule and 100 fg/ml to 10 ng/ml for the BRET2 biosensor variant.
This study provides evidence that VEGF can be quantified using a single molecule enabling BRET over a wide concentration range by changing the conformation of the VEGF binding domain of the sensor by antigen (VEGF) interaction.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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