Vascular Endothelial Growth Factor (VEGF) Concentration Is Underestimated by Enzyme-Linked Immunosorbent Assay in the Presence of Anti-VEGF Drugs

PURPOSE. Commercially available enzyme-linked immunosorbent assay (ELISA) kits are often used to monitor vascular endothelial growth factor (VEGF) levels in exudative age-related macular degeneration. To test their accuracy, this study performed measurements using the ELISA kits in the presence of anti-VEGF drugs. METHODS. The concentrations of bevacizumab, pegaptanib, or ranibizumab at 28 days and aflibercept at 28 and 56 days after an injection were estimated based on previous pharmacokinetic studies. Vascular endothelial growth factor concentrations were measured with two widely used VEGF ELISA kits in the presence of anti-VEGF drugs or control mouse immunoglobulin G (IgG). The monocyte chemotactic protein-1 (MCP-1) ELISA kit was used as a non-VEGF ELISA control kit. RESULTS. The concentrations of aflibercept, bevacizumab, pegaptanib, and ranibizumab were estimated at 0.14 to 7.2, 4.9, 8.6, and 0.11 to 1.1 mu g/mL, respectively. ELISA underestimated the VEGF concentration 2- to 100-fold lower in the presence of an anti-VEGF drug, except for pegaptanib, at all VEGF concentrations tested (7.8-1500 pg/mL). Vascular endothelial growth factor at 1000 pg/mL was measured as 92, 150, and 170 pg/mL in the presence of aflibercept (7.2 mu g/mL), bevacizumab (4.9 mu g/mL), and ranibizumab (1.1 mu g/mL), respectively (all P < 0.0001), and the measured VEGF concentration decreased proportionately by 90% to 92% with aflibercept, 85% to 94% with bevacizumab, and 83% to 99% with ranibizumab. The control mouse IgG did not interfere with the measurement of VEGF. Ranibizumab did not affect the measurements with MCP-1 ELISA. CONCLUSIONS. Investigators should exercise caution when interpreting measurements of VEGF ELISA in patients being treated with an anti-VEGF drug.