Anti-VEGF Drug Interference in the R&D Systems Quantikine VEGF-A ELISA Kit

摘要

Purpose : To examine the impact of anti-VEGF drugs on the quantitation of VEGF by the R&D Systems Quantikine VEGF-A ELISA kit. Methods : Surface Plasmon Resonance (SPR) experiments were performed using a Biacore 3000 instrument to determine the relative binding affinities of the anti-VEGF drugs and the Quantikine VEGF-A capture antibody. Aflibercept and bevacizumab were captured on a coupled Protein A chip surface, while ranibizumab was captured on a coupled anti-human Fab polyclonal antibody chip surface. The Quantikine VEGF-A capture antibody was captured on a coupled anti-mouse Fc?3 chip surface. Following the capture step, varying concentrations of test ligand were individually injected over the surfaces. The Quantikine VEGF-A ELISA was performed as per manufacturera??s instructions. Separate samples containing 1.3 pM (50 pg/mL) of VEGF were prepared with increasing concentrations of each of the 3 anti-VEGF inhibitors. Results : The binding constant (KD) of the Quantikine VEGF-A capture antibody for VEGF was 1.41 pM, which fell between the KD of aflibercept (0.490 pM) and the KDs of ranibizumab (46 pM) and bevacizumab (58 pM). Aflibercept, ranibizumab, and bevacizumab all interfered with the measurement of VEGF concentrations using the Quantikine VEGF-A ELISA. However, the IC50 for aflibercept (0.45 pM) was much lower than the IC50 values of either ranibizumab (24 pM) or bevacizumab (138 pM). The observed degree of interference correlated with the relative binding affinities of the 3 anti-VEGF inhibitors for VEGF. Conclusions : Aflibercept, ranibizumab and bevacizumab all caused assay interference in the Quantikine VEGF-A ELISA. The level of assay interference was proportional to the binding affinities of each anti-VEGF inhibitor for VEGF. Aflibercept, with the highest binding affinity for VEGF A165, interfered with the detection of VEGF by the Quantikine VEGF-A ELISA to a greater degree than either ranibizumab or bevacizumab. However, serum concentrations of each of these drugs following IVT administration all reach circulating levels that have the potential to negatively affect accurate measurement of VEGF levels. This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.