泰它西普免疫原性分析方法建立及验证

Development and validation of immunogenicity methods for detecting telitacicept

目的:建立桥式酶联免疫吸附法测定食蟹猴血清中抗药抗体(anti-drug antibody, ADA)和竞争酶联免疫吸附法测定中和抗体(neutralizing antibody, NAb),并进行方法学验证。方法:桥式酶联免疫吸附法在96孔板预包被泰它西普(代号:RC18),与待测样品中的抗RC18抗体结合形成复合物,依次加入生物素化的RC18(Biotin-RC18)、辣根过氧化物酶标记链霉亲和素(SA-HRP)和四甲基联苯胺底物(TMB)显色,终止反应后在酶标仪450 nm/630 nm波长处读取吸收度。竞争酶联免疫吸附法在96孔板预包被B细胞激活因子或增殖诱导配体蛋白,加入与Biotin-RC18预混合的样品,形成B细胞激活因子或增殖诱导配体-抗RC18抗体-Biotin-RC18三者的复合物,依次加入SA-HRP和TMB显色,终止反应后在酶标仪上读取吸收度。结果:桥式酶联免疫吸附法线性范围的精密度≤12.32%,灵敏度为50 ng·mL^(-1),筛选临界阈值为0.937,确证临界阈值为23.62%。竞争酶联免疫吸附法方法线性范围的精密度≤20%,灵敏度为312.50 ng·mL^(-1),针对靶标B细胞激活因子和增殖诱导配体的NAb活性,判断阈值分别为0.79和0.69,可耐受血清中RC18的药物浓度分别为2.5和5μg·mL^(-1)。结论:方法学验证结果表明,桥式酶联免疫吸附法及竞争酶联免疫吸附法均符合临床前生物制品免疫原性研究的要求,可用于食蟹猴血清中ADA及ADA阳性样本NAb的检测。

Objective:To establish a bridging enzyme-linked immunosorbent assay(ELISA)method for the de⁃termination 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 in⁃ducing 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 re⁃spectively can tolerate 2.5μg·mL^(-1) and 5μg·mL^(-1) RC18 in serum.Conclution:The results of method vali⁃dation indicate that both bridging ELISA and competitive ELISA meet the requirements of preclinical immunoge⁃nicity studies of biological products,and can be used for analysis of the concentrations of ADA and NAb in cyno⁃molgus monkey serum.