Objective: To establish a bridging enzyme-linked immunosorbent assay (ELISA) method for the determination of anti-drug antibody(ADA) and a competitive ELISA method for the determination of neutralizing antibody(NAb)in cynomolgus monkey serum, and to conduct methodological validation. Methods: The steps of bridging ELISA method were as follows: the 96-well plates were precoated with telitacicept(RC18) which could combine with anti-RC18 antibody in the samples to form a complex, then were sequentially added biotinylated RC18(Biotin-RC18), horseradish peroxidase conjugated streptavidin (SA-HRP), and tetramethylbenzidine (TMB) substrate solution for color development. After terminating the reaction, the absorbance was read at a wavelength of 450 nm/630 nm on an ELISA reader. The procedures of competitive ELISA method were as follows: the 96-well plates were precoated with B-cell activation factor of the TNF family (BAFF) or a proliferation inducing ligand (APRIL) protein and then were added the samples which was pre-mixed with Biotin-RC18 to form BAFF or APRIL anti-RC18-antibody and Biotin-RC18 complex. SA-HRP and TMB substrate solution for color development were added sequentially. After terminating the reaction, the absorbance was read at an ELISA reader with dual wave length. Results: The precision of linear range of bridging ELISA method was less than 12.32%, the sensitivity was 50 ng·mL-1, the critical threshold of screening was 0.937, and the critical threshold of confirmation was 23.62%. The precision of the linear range of competitive ELISA method was less than 20%, the sensitivity was 312.50 ng·mL-1, and the threshold for determining the activity of NAb against the target BAFF and APRIL was 0.79 and 0.69, respectively. On BAFF and the method of research targets respectively can tolerate 2.5 μg·mL-1 and 5 μg·mL-1 RC18 in serum. Conclution: The results of method validation indicate that both bridging ELISA and competitive ELISA meet the requirements of preclinical immunogenicity studies of biological products, and can be used for analysis of the concentrations of ADA and NAb in cynomolgus monkey serum.
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