Objective： To establish an enzyme linked immunosorbent assay（ ELISA） method for determination of anti-CTLA-4 monoclonal antibody（ ipilimumab,YERVOY®） in Cynomolgus monkey serum. Methods： Anindirect ELISA method was established for the determination of the monoclonal antibody drug. Human CTLA- 4 protein was coated in microplate. After plate blocking,diluted samples were added. Then HRP labeled Goat Anti-human IgG antibody was used as enzyme-labeled antibody. TMB substrate was added for color development and the reaction was terminated by 2 mol·L-1 of sulfuric acid. Finally,the absorbance of each well was measured by a microplate reader at 450/630 nm. The calibration curve was generated by the D450/630 nm value（ Y） versus the concentration（ X）.A 5-parameter logistics algorithm was used to get the standard curve equation,and the sample D450/630 nm to be tested was substituted into the equation to obtain the sample concentration to be tested. Results： Under the optimized conditions,the method dynamic range was 0.625-30.000 μg·mL-1,with the lowest quantification limit of 0.625 μg·mL-1. Precision（ within-plate and between plate） was within ±15% and the accuracy ranged from 75% to 125%. The specificity,selectivity and dilution linearity of the method all met the acceptable standard,and the accuracy ranged from 80% to 120%. At the same time,the anti-CTLA-4 monoclonal antibody was stable in room temperature,freeze-thaw,and long-term stability tests in Cynomolgus monkey serum meanwhile,and the sample accuracy ranged from 80% to 120%. Conclusion： The method established in this study meets the requirements of the guidelines for verification of quantitative analysis methods for biological samples in the Pharmacopoeia of the People’s Republic of China 2020 edition. It is proven to be feasible for the quantitative analysis of anti-CTLA-4 monoclonal antibody（ YERVOY®） in Cynomolgus monkey serum.
LI Qing-lian, WANG Bin, XU Nan-nan, WANG Ning, SUN Yun-xia, LI Kai-tong
. Development of an enzyme linked immunosorbent assay method for the determination of recombinant anti CTLA-4 monoclonal antibody in Cynomolgus monkey serum[J]. Chinese Journal of Pharmaceutical Analysis, 2021
, 41(7)
: 1209
-1217
.
DOI: 10.16155/j.0254-1793.2021.07.12
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