August 2018
Volume 59, Issue 10
Open Access
Letters to the Editor  |   August 2018
Author Response: Comments on Assays Used to Measure VEGF in the Presence of Anti-VEGF Therapeutics
Author Affiliations & Notes
  • Hidenori Takahashi
    Jichi Medical University, Shimotsuke-shi, Tochigi, Japan; and
  • Yasuo Yanagi
    Medical Retina, Singapore National Eye Centre, Singapore Eye Research Institute, Eye-ACP, Duke NUS Medical School, National University of Singapore, Singapore.
Investigative Ophthalmology & Visual Science August 2018, Vol.59, 4107. doi:10.1167/iovs.18-25216
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      Hidenori Takahashi, Yasuo Yanagi; Author Response: Comments on Assays Used to Measure VEGF in the Presence of Anti-VEGF Therapeutics. Invest. Ophthalmol. Vis. Sci. 2018;59(10):4107. doi: 10.1167/iovs.18-25216.

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      © ARVO (1962-2015); The Authors (2016-present)

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We wish to thank the authors for their letter1 and comments on our paper.2 In our paper, we clearly pointed out VEGF concentration is underestimated by ELISA assay in the presence of anti-VEGF drugs and suggested that investigators should exercise caution when interpreting measurements of VEGF ELISA in patients being treated with an anti-VEGF drug. The authors concur with us. In a recent paper the authors cited, one of the authors states, “as noted by the manufacturers of this kit, this assay (i.e., ELISA assay kit they used for their study) is not suited to measure total VEGF in the presence of anti-VEGFs, because of the binding properties of the capture and detection reagents used in the assay. However, this assay is commonly used for measuring free-VEGF levels in samples from patients treated with anti-VEGF therapeutics.”3 
The authors point out the importance of measuring “free” or unbound VEGF concentrations. In our IOVS paper, we also implied that ELISA does not measure the free-VEGF concentrations in the presence of anti-VEGF antibody either. The authors mention that literature-derived Kd values we used may not be appropriate to derive free-VEGF concentrations. However, neither their paper4 nor our paper support the usefulness of ELISA assays in assessing the level of unbound VEGF in the presence of anti-VEGF drugs (to assist in the assessment of their therapeutic and safety impacts). 
The paper by Yang et al.4 used a Biacore system to evaluate the relative binding and potencies of three inhibitors of VEGF, used to treat neovascular AMD, and assessed their relevance in the context of clinical outcome. They concluded that aflibercept did not bind with higher affinity than ranibizumab to VEGF. We agree that we need to be careful in interpreting data on biomolecular interactions. The variability of Kd may be due to various factors, such as the assay format used. We actually noted that other studies do not support their results.5,6 The authors also mention that VEGF/anti-VEGF complexes in the samples diluted into an assay will tend to dissociate and have more free-VEGF, and therefore, assume that the ELISA assays will tend to overestimate free-VEGF levels. However, this assumption is incorrect, because anti-VEGF drugs in the samples may interfere with VEGF ELISA measurements (see Fig. 1 in our original article2) and ELISA assays do not measure the concentration of “free” or unbound VEGF. This is clearly mentioned in our original article. There is also a possibility that ELISA detects VEGF/anti-VEGF complex. 
Therefore, for now, we believe it would be prudent to consider measured concentration is not the same as the free-VEGF concentrations. Further studies are needed to address whether ELISA assays are really useful in assessing “free"-VEGF levels in the presence of anti-VEGF drugs. Until then, clinicians should exercise caution when interpreting measurements of VEGF ELISA in patients being treated with an anti-VEGF drug. 
References
Wakshull E, Avery R, Maia M. Comments on assays used to measure VEGF in the presence of anti-VEGF therapeutics. Invest Ophthalmol Vis Sci. 2018; 59: 4106.
Takahashi H, Nomura Y, Nishida J, Fujino Y, Yanagi Y, Kawashima H. Vascular endothelial growth factor (VEGF) concentration is underestimated by enzyme-linked immunosorbent assay in the presence of anti-VEGF drugs. Invest Ophthalmol Vis Sci. 2016; 57: 462–466.
Avery RL, Castellarin AA, Steinle NC, et al. Systemic pharmacokinetics and pharmacodynamics of intravitreal aflibercept, bevacizumab, and ranibizumab. Retina. 2017; 37: 1847–1858.
Yang J, Wang X, Fuh G, et al. Comparison of binding characteristics and in vitro activities of three inhibitors of vascular endothelial growth factor A. Mol Pharm. 2014; 1: 3421–3430.
Walker A, Chung CW, Neu M, et al. Novel interaction mechanism of a domain antibody-based inhibitor of human vascular endothelial growth factor with greater potency than ranibizumab and bevacizumab and improved capacity over aflibercept. J Biol Chem. 2016; 291: 5500–5511.
Papadopoulos N, Martin J, Ruan Q, et al. Binding and neutralization of vascular endothelial growth factor (VEGF) and related ligands by VEGF Trap, ranibizumab and bevacizumab. Angiogenesis. 2012; 15: 171–185.
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