ELISA法检测人血清中重组人信号调节蛋白α-抗CD20人鼠嵌合抗体融合蛋白IMM0306的方法学建立和验证  

Development and validation of ELISA method for detection of human signal regulatory protein α-anti-CD20 mouse chimeric antibody fusion protein IMM0306 in human serum

作　　者：靖钰 姚慕蓉 李松 陈典泽 张力 杨勇 钟勘 宗山海 JING Yu;YAO Mu-rong;LI Song;CHEN Dian-ze;ZHANG Li;YANG Yong;ZHONG Kan;ZONG Shan-hai(HQ Bioscience Co.,Ltd.,Suzhou 215000,China;ImmuneOnco Biopharma Co.,Ltd.,Shanghai 201200,China)

机构地区：[1]苏州海科医药技术有限公司,江苏苏州215000 [2]宜明昂科生物医药技术(上海)有限公司,上海201200

出　　处：《药学学报》2021年第9期2367-2371,共5页Acta Pharmaceutica Sinica

摘　　要：IMM0306是重组人信号调节蛋白α-抗CD20人鼠嵌合抗体融合蛋白,拟在临床中治疗难治或复发性CD20阳性B细胞非霍奇金淋巴瘤(B-NHL)。本文建立了ELISA法(酶联免疫吸附法)用于评价人血清中IMM0306的药代动力学。该实验获得中国医学科学院肿瘤医院伦理委员会批准(批准号:CTR20192612)。用重组人CD47蛋白包被过夜, 5%脱脂奶粉封闭2 h,洗涤板后,按照板位图每孔加入100μL样本孵育1.5 h,洗涤后加入生物素标记的检测抗体anti-IMM0306-biotin并孵育1 h,随后加入辣根过氧化物酶(HRP)标记的链霉亲和素孵育1 h,显色并检测。捕获试剂的最佳包被浓度为2μg·mL^(-1),检测试剂anti-IMM0306-biotin和链霉亲和素-HRP的最佳稀释度分别为1∶500和1∶5 000。该方法的定量下限为4 ng·mL^(-1),标准曲线范围是4~100 ng·mL^(-1)。方法的验证结果均满足相应的接受标准,可以用于IMM0306临床药代动力学研究。IMM0306 is a recombinant human signal regulatory protein α-anti-CD20 mouse chimeric antibody fusion protein, intended to treat refractory or recurrent CD20 positive B-cell non-Hodgkin lymphoma(B-NHL) in clinical. In this study, an ELISA method was established to evaluate the pharmacokinetics of IMM0306 in human serum. The experiment was approved by the Ethics Committee of the Cancer Hospital of the Chinese Academy of Medical Sciences(No. CTR20192612). Recombinant human CD47 protein was coated with the plate overnight,blocking the plate with 5% skin milk for 2 h. After washing, 100 μL per well standard and unknown samples were added, and incubated for 1.5 h. After washing, the detection antibody Anti-IMM0306-Biotin was added and incubated for 1 h, and then HRP-labeled streptavidin was added for 1 h, and the color was detected. The optimal concentration of coating reagent was 2 μ g ·mL^(-1) by ELISA method, and the optimal dilution of anti-IMM0306-biotin and SA-HRP were 1∶500 and 1∶5 000, respectively. The lower limit of quantitation was 4 ng·mL^(-1), and the standard curve range was 4-100 ng·mL^(-1). The verification results of the method meets the corresponding acceptance criteria, and can be used in IMM0306 clinical pharmacokinetic studies.

关 键 词：CD47 抗体融合蛋白 ELISA 药代动力学 

分 类 号：R9I7[医药卫生—药学]