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Clinical Trial
. 2016 Oct 11:6:34631.
doi: 10.1038/srep34631.

Detection of aqueous VEGF concentrations before and after intravitreal injection of anti-VEGF antibody using low-volume sampling paper-based ELISA

Min-Yen Hsu  1   2   3   4 , Yu-Chien Hung  1 , De-Kuang Hwang  1   5 , Shang-Chi Lin  2 , Keng-Hung Lin  1 , Chun-Yuan Wang  1 , Hin-Yeung Choi  6 , Yu-Ping Wang  7 , Chao-Min Cheng  8
Affiliations
Clinical Trial

Detection of aqueous VEGF concentrations before and after intravitreal injection of anti-VEGF antibody using low-volume sampling paper-based ELISA

Min-Yen Hsu et al. Sci Rep. .

Abstract

Intraocular vascular endothelial growth factor (VEGF) levels play an important role in the pathogenesis of blindness-related diseases, such as age-related macular degeneration (AMD). Here, we aimed to develop a paper-based enzyme-linked immunosorbent assay (P-ELISA) to analyze the suppression of aqueous VEGF concentrations following intravitreal injection (IVI) of anti-VEGF antibody (bevacizumab or ranibizumab). A total of 25 eyes with wet AMD, one with myopic neovascularization, and one with polypoidal choroidal vasculopathy were enrolled in this study. The limit of detection using P-ELISA was 0.03 pg/mL. Forty-six consecutive samples of aqueous humor were acquired. From all samples, 66.67% (10/15) achieved complete VEGF suppression (below the detection limit) within 5 weeks of receiving IVI of anti-VEGF antibody. Only 13.33% of samples (2/15) achieved complete VEGF suppression 5 weeks after receiving treatment. In some patients, elevated VEGF was still detected 5 weeks after receipt of anti-VEGF antibody, and all samples (10/10) were found to have elevated VEGF levels 49 days after treatment. Thus, we suggest that monthly IVI of anti-VEGF antibody may be required to ensure durable VEGF inhibition. Ultrasensitive P-ELISA can detect elevated VEGF at an earlier time point and may facilitate decision-making regarding appropriate treatment strategies.

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Conflict of interest statement

M.-Y.H., K.H.L., and C.M.C. are inventors of a patent (patent no.: TW 201428105) owned by National Tsing Hua University based on the work reported in this paper. M.-Y.H. and C.M.C. have a patent pending in the USA based on the work reported in this paper (patent no:US20140315224 A1). Y.-C.H., D.-K.H., S.-C.L., K.-H.L., C.-Y.W., Y.-P.W. and H.-Y.C. have no potential conflicts of interest to declare.

Figures

Figure 1
Figure 1. Calibration curve of paper-based ELISA for VEGF detection.
We used a commercial kit for VEGF labeling at different concentrations ranging from 0.01 to 105 pg/mL.
Figure 2
Figure 2. Schematic illustration of paper-based ELISA for VEGF detection.
In the clinical setting, patients receive standard-of-care procedures: slit lamp, SD-OCT, and FAG (left panel). Paper-based ELISA requires 1 μL of aqueous humor before operation, and quantification of VEGF concentrations requires 40 μL (right panel). Further colorimetric results can be obtained using a scanner or smartphone. Ophthalmologists can determine treatment according to VEGF concentrations and SD-OCT results.
Figure 3
Figure 3. Aqueous VEGF levels before and after intravitreal injection of anti-VEGF antibody.
Blue dots represent VEGF levels before IVI (group 1). Black dots represent VEGF concentrations after IVI. The horizontal red dotted line is the detection limit of paper-based ELISA, approximately 0.03 pg/mL. The horizontal black dashed line is the detection limit of conventional ELISA or Luminex, approximately 4–5 pg/mL. The earliest elevation of VEGF was found in a patient with PCV at 3 weeks after IVI (arrow). Four earlier elevations of VEGF levels were found 5 weeks after IVI injection by ultrasensitive paper-based ELISA (asterisk).
Figure 4
Figure 4. VEGF levels before and after IVI. Group 1: pre-IVI samples; group 2: samples collected within 5 weeks of IVI; group 3: samples collected more than 5 weeks after IVI.
Figure 5
Figure 5
(a) Analysis of VEGF concentrations in patients receiving bevacizumab. (b) Analysis of VEGF concentrations in patients receiving ranibizumab.
Figure 6
Figure 6. Schematic comparison of routine follow-up strategies and follow-up based on analysis of VEGF concentrations.
In the routine strategy, most of the nonresponders can be identified after several anti-VEGF injections several months later, during which time devastating, irreversible vision loss can occur. Based on P-ELISA or other point-of-care (P-O-C) diagnostics, VEGF concentrations within the eye can provide the ophthalmologist with valuable information and facilitate decision-making regarding treatments options.
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