ELISA法测定恒河猴血清中艾塞那肽衍生物的抗药抗体  

Detection of anti-drug antibody against exenatide derivant in Rhesus monkey serum

作　　者：宋海晶[1] 徐静芝 董斌 董立厚[3] 邹佳[3] 王清清[3] 欧伦[3] 孙效[4] 陈方[3] 

机构地区：[1]解放军第306医院急诊部,北京100105 [2]北京和实康明医药科技有限公司,北京102206 [3]军事医学科学院放射与辐射医学研究所,北京100850 [4]解放军总后勤部丰台综合仓库药材供应站,北京100071

出　　处：《中国新药杂志》2013年第19期2256-2261,共6页Chinese Journal of New Drugs

摘　　要：目的:建立测定恒河猴血清中艾塞那肽衍生物抗药抗体(ADA)的ELISA方法并进行验证,用于测定该药的抗药抗体。方法:将药物包被在96孔板中作为捕获试剂,捕获猴血清样本中的抗药抗体,羊抗猴IgG辣根过氧化物酶偶联二抗作为检测试剂与被捕获的抗药抗体结合。阴性对照孔加入的血清样本为经筛选的空白基质,而阳性对照孔直接包被经筛选的空白恒河猴血清,其中的IgG被包被于板孔中,其余步骤与阴性对照孔一致。方法建立后进行筛选判断阈值,确证判断阈值,方法精密度验证。结果:筛选判断阈值为0.1,方法批内批间精密度小于25%,样品复孔A值变异小于15%,确证判断阈值为84%游离药物抑制率。艾塞那肽衍生物连续给药13周后在恒河猴体内产生抗药抗体,抗体阳性率与对照药相当。结论:成功建立及验证该方法,并用于艾塞那肽衍生物长期毒性研究伴随免疫原性测定中,成为该药申请临床试验的报批资料之一。Objective： To develop and validate an ELISA method for the determination of anti-drug anti- body against exenatide derivant in Rhesus monkey serum. Methods： The drug was coated on the plate as capture reagent, and then the anti-drug antibody in Rhesus monkey serum was captured by the drug. Next a goat-anti-mon- key IgG-HPR conjugate bound to the captured drug as detection reagent. The non-screened, pooled blank matrix was added in the well instead of samples as negative control. Positive control had no capture step that non-specific IgG in the Rhesus monkey serum was directly coated on the well. The other steps were the same as those of samples and negative. After the method was developed, validation of screening cut point, precision and inhibition rate cut point were conducted. Results： The threshold titer of screening was 0.1, inter- and intra-batch precisions were not more than 25% , and the variation of duplicate A values was less than 15%. The exenatide derivant could trigger anti-drug antibody in Rhesus monkeys when they were consecutively dosed for 13 weeks. The positive rate was similar as original exenatide. Conclusion： The method is validated and successfully applied in the long term toxicity studycompanied immunogenicity evaluation. The results have been one part of the application files for clinical trial.

关 键 词：抗药抗体 免疫原性 艾塞那肽衍生物 酶联免疫吸附分析 

分 类 号：R977.15[医药卫生—药品]