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Hidenori Takahashi, Yujiro Fujino, Yasuo Yanagi; Errors In Measuring VEGF Concentrations In The Presence Of Anti-VEGF Antibodies By Using ELISA. Invest. Ophthalmol. Vis. Sci. 2012;53(14):6289.
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Several reports have investigated the intraocular concentration of vascular endothelial growth factor (VEGF) after administration of anti-VEGF antibodies. Enzyme-linked immunosorbent assay (ELISA), which is widely used for measuring VEGF concentration, uses specific antibodies. Therefore, if anti-VEGF antibodies are already present in the samples, competition between these antibodies would introduce errors in measuring the concentration of VEGF in the samples. To confirm these hypothesis, we measured VEGF concentration in the presence of anti-VEGF antibody by using ELISA.
We used the R & D Systems Quantikine Human VEGF ELISA kit (Minneapolis, MN, USA) for this study. Ranibizumab, bevacizumab, pegaptanib, and mouse control immunoglobulin G (IgG) were added to 1000, 500, 250, 125, 63, 31, 16, and 7.8 pg/ml VEGF. Using the previously investigated half-life of these compounds, we calculated the each antibody concentration in the vitreous cavity 28 days after the antibody injection, which showed that the concentration of ranibizumab, bevacizumab, pegaptanib, and IgG was calculated as 0.11, 13, 8.6, and 13 μg/ml respectively.
VEGF was measured at 540, 290, 120, 58, 29, 12, 6.4 pg/ml and undetectable in the presnce of ranibizumab. VEGF was measured at 31 and 10 pg/ml and undetectable from then on in bevacizumab group. VEGF was measured at 1000, 480, 230, 110, 55, 22, 11 pg/ml and undetectable in pegaptanib group. VEGF was measured at 1000, 650, 300, 150, 73, 32, 13, and 7.2 pg/ml in IgG group.
The VEGF concentration measured by ELISA was lower than the total VEGF concentration when anti-VEGF antibodies were present. It was much lower when the antibodies had a low dissociation constant or when there was a high concentration of antibodies. Caution should be exercised when interpreting the VEGF concentration measured by ELISA in the presence of anti-VEGF antibody.
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