摘要
目的:建立运用Bridging-ELISA检测食蟹猴血清中抗体偶联药物(RC48)抗药抗体的方法,用于检测食蟹猴血清中的抗药物抗体。方法:采用Bridging-ELISA法进行检测,96孔板预先包被RC48,与待测样品中的抗RC48抗体结合形成复合物,依次加入偶联Biotin的RC48和HRP标记的亲和素和底物TMB显色,用酶标仪读取A_(450nm)值,参比波长630 nm;阴性空白对照样品为食蟹猴混合空白血清;阳性对照样品为分别针对MMAE单抗和针对裸抗体多抗的混合抗体。结果:建立了检测抗RC48抗体的Bridging-ELISA法,并进行了方法学验证。确定方法线性范围的精密度CV%≤19.0,灵敏度为85.48 ng·m L^(-1),筛选临界阈值为1.33,确证临界阈值为67.2%,系统适用性和药物耐受性良好。结论:方法验证结果均符合临床前生物制品免疫原性研究的要求,已用于非临床研究中食蟹猴血清中抗RC48抗体的检测。
Objective： To develop and validate a Bridging-ELISA method for determination of anti-drug antibody against an antibody drug conjugate（ RC48） in cynomolgus monkey serum. Methods： The drug（ RC48）was coated on the plate as a capture reagent,and then the anti-drug antibody in cynomolgus monkey serum was captured by the drug. Next,Biotin-conjugated RC48 bound to the captured anti-drug antibody as a bridging detection reagent. The pooled normal serum from non-immunized cynomolgus monkeys was added in the well as the negative control. Positive control was the mixture of anti-MMAE monoclonal antibody and anti-RC48 polyclonal anti body. Results： The Bridging-ELISA method was established and verified for the determination of anti-RC48 antibody. The threshold titer of screening was 1. 33,specificity cut point was 67. 2%,inter- and intra-batch precisions were not more than 19. 0%,and sensitivity was 85. 48 ng·m L- 1. The method had a high suitability,and drug tolerance was well. Conclusion： The method validation results conform to the preclinical immunogenicity requirements of biotechnology products. The method has already been used as detection of anti-RC48 antibody in cynomolgus monkey serum.
出处
《中国新药杂志》
CAS
CSCD
北大核心
2016年第6期639-644,共6页
Chinese Journal of New Drugs
基金
国家"重大新药创制"科技重大专项(2014ZX09508004)
