基于抗CD20单克隆抗体的2种夹心ELISA法的建立  

Development of Two Sandwich Enzyme Linked Immunosorbent Assays for Determination of Anti-CD20 Monoclonal Antibody

作　　者：邓承莲[1,2] 董立厚[2] 邹佳[2] 宋海峰[2] 

机构地区：[1]广西医科大学,广西南宁530002 [2]军事医学科学院放射与辐射医学研究所,军事医学科学院药代动力学重点实验室,北京100850

出　　处：《生物技术通讯》2014年第3期406-409,共4页Letters in Biotechnology

基　　金：国家十一五;十二五重大新药创制科技重大专项(2009ZX09304-004;2012ZX09301003-001-007)

摘　　要：目的：建立和比较2种灵敏、特异的夹心ELISA方法，用于准确定量检测食蟹猴血浆中重组抗CD20人源化单克隆抗体（rh—anti—CD20zumab）浓度。方法：以rh—anti—CD20zumab为基础，分别用山羊抗人IgGF（ab＇）2抗体和驴抗人IgG Fc抗体包被96孔酶标板，加入待测样品，均采用HRP标记的猴血清吸附的山羊抗人IgG抗体进行检测，加底物显色，读取D450nm值。结果：建立了2种定量检测rh—anti—CD20zumab的夹心ELISA方法并进行了确证，样品的前处理分别为1：20和1：10，方法的线性范围分别为40～5000和40～12500ng／mL，定量下限均为40ng／mL，两者的板内、板间精密度分别小于16.2％和19．4％，准确度分别为-10．3％～16．6％和-14．4％～12．9％。2种方法均具有良好的特异性和稀释线性，且都未出现钩状效应。结论：方法学确证表明，本研究建立的2种ELISA法均符合新生物制品临床前药代动力学研究指导原则的要求，可用于rh—anti—CD20zumab的检测，为后续rh—anti—CD20zumab在食蟹猴体内的药代动力学研究提供了不同的检测方法。Objective： To establish two kinds of sensitive and specific sandwich ELISA methods for accurate quantification of recombinant anti-CD20 humanized monoclonal antibody（rh-anti-CD20zumab） in cynomolgus monkey plasma and compared with each other. Methods： Based on rh-anti-CD20zumab, goat anti-human IgG F（ab＇）2 antibody and donkey anti-human IgG Fc antibody were used to coat in 96-well microtiter plate respectively. After the samples were added, HRP-labeled and monkey-absorbed goat anti-human IgG antibody was added to detect, then color was developed by adding substrate solution and the reaction was stopped by added stop solution. Final- ly the plate was read at a wavelength of 450 nm by using a microplate reader. Results： Two kinds of sandwich ELISA methods were developed to determine the concentration of rh-anti-CD20zumab and confirmed subsequently. For the two methods, the pre-treatments of sample were 1：20 and 1：10, and the linear ranges were 40-5000 and 40-12 500 ng/mL respectively. The lowest quantification of two methods were both 40 ng/mL. The preeisions of intra-day and inter-day were both 〈16.2% and 〈19.4%, the aceuracys were -10.3%-16.6% and -14.4%-12.9%, respectively. Both of them showed good specificity and dilutional linearity, and no hook effect was occurred in two methods. Conclusion： The methodology validation confirmed that both of these two methods established in this study were in line with the guidelines of preclinical pharmacokinetic study of new biological products, which can be used to determine the concentration of rh-anti-CD20zumab and provided different methods for pharmacokinetic studies of rh-anti-CD20zumab in cynomolgus monkeys subsequently.

关 键 词：CD20 单克隆抗体 酶联免疫吸附法 药代动力学 

分 类 号：R392.1[医药卫生—免疫学]