[Establishment and optimization of an ELISA for affinity detection of single-chain antibodies to EGFRvIII]

Objective To establish an ELISA for the detection of the affinity of the epidermal growth factor receptor variant III (EGFRvIII ) single-chain antibodies PD0721, and optimize the experimental conditions. Methods An indirect ELISA for the detection of the affinity of the PD0721 single-chain antibody which was prepared by our laboratory was established. By square matrix titration method of indirect ELISA, the experimental conditions including antibody and antigen concentrations and coating conditions were optimized, and finally the sensitivity and precision of the method were analyzed. Results The antigen was diluted with PBS to 1.25 mg/L and coated at 4 DegreesCelsius for 12 hours, 120 ng/mL PD0721 single-chain antibody and enzyme-labeled antibody at the dilution of 1:8000 were added for the best results. Indirect ELISA exhibited high performance within a dynamic range 15 ng/mL-480 ng/mL, and the limit of detection (LOD) was 7.5 ng/mL. The intra-assay coefficient variations (CV) ranged from 0.11% to 0.99% and inter-assay CV ranged from 0.68% to 3.15%. Conclusion An accurate and stable ELISA for detecting the affinity of PD0721 single-chain antibody has been established, which laid a foundation for future preparation of antibody-conjugated drugs.