ELISA法测定大鼠血清中聚乙二醇化重组蛋白(PEG-SOP55)浓度及药代动力学研究  

Determination of PEGylated recombination protein(PEG-SOP55) in rat serum by ELISA and study of its pharmacokinetics

作　　者：张涛[1] 何静[1] 李坤[1] 王明蓉[1] ZHANG Tao HE Jing LI Kun WANG Ming-rong(Chengdu Institute of Biological Products Co., Ltd., Cllengdu 610023, China)

机构地区：[1]成都生物制品研究所有限责任公司,成都610023

出　　处：《药物分析杂志》2016年第10期1767-1777,共11页Chinese Journal of Pharmaceutical Analysis

摘　　要：目的:建立一种检测大鼠血清中聚乙二醇化重组蛋白(PEG-SOP55)浓度的ELISA方法,并进行该蛋白的药代动力学初步研究。方法:以该蛋白的多抗包被,标记生物素的单抗检测,生物素-链霉亲和素-辣根过氧化物酶两级酶联放大检测信号,构建双抗夹心ELISA法进行浓度检测。通过对包被抗体和检测抗体工作浓度的优化实验,血清基质的干扰实验,不同结构聚乙二醇分子的比较实验,建立该方法的基本实验条件。并参考相关法规,对其方法特异性、准确度、精密度、耐用性、检测限度以及样品稳定性进行验证。分别用PEG-SOP55_(30kDa)、PEG-SOP55_(40kDa)、PEG-SOP55_(40kDa branched)进行Lewis大鼠腹腔单次给药然后测定血清浓度,获得该蛋白用不同分子结构聚乙二醇分子修饰后的药代动力学参数。结果:实验证实聚乙二醇修饰降低了检测抗体和目标分子的结合效率,且大鼠血清和聚乙二醇分子结构变化对检测有干扰。通过提高包被抗体浓度,获得对目标分子较好的捕获效率。通过将血清样品稀释液从PBS替换为咪唑缓冲液,且保持标准曲线和待测样品血清稀释倍数一致,克服了血清基质所带来的干扰。通过保持标准曲线和待测样品聚乙二醇分子结构一致,克服了因聚乙二醇分子结构不同带来的干扰。方法验证结果表明该方法特异性良好,回收率为(100±15.2)%,实验内精密度和实验间精密度验证数据RSD均不超过20%,不同操作人员对检测结果无显著影响,标准曲线线性良好,标准曲线的最高和最低浓度能够准确测定。将大鼠血清样品反复冻融3次,在室温放置4 h其稳定性均符合要求,满足该方法检测条件。通过对Lewis大鼠血清样品进行浓度测定,获得PEG-SOP55_(30kDa)、PEG-SOP55_(40kDa)、PEG-SOP55_(40kDa branched)的药代动力学参数。其C_(max)分别为31.9、54.4、67.7 mg·m L^(-1),AUC_(0-∞)分别为823.0、1 978.0、3 502.6 mg·h·m L^Objective：To develop an ELISA method for content determination of a PEGylated recombinant protein（PEG-SOP55）in rat serum,and to study its pharmacokinetic features in Lewis rat.Methods：A sandwich enzyme linked immunosorbent assay（ELISA）method was adopted.Briefly,the plates were coated with the SOP55 polyclonal antibody,detected with the biotin labeled monoclonal antibody using ＂biotin-streptavidin-horseradish peroxidase＂ two-stage enzyme cascade to amplify detection signal,named 3E3-biotin.The experimental conditions were established by optimizing the working concentration of the coating antibody and detection antibody,reducing the interference of serum matrix,and minimizing the interference caused by the difference of the polyethylene glycol molecule structures.With reference to the relevant regulations,the method had been validated for its specificity,accuracy,precision,robustness,detection range and sample stability.Single intraperitoneal administration of PEG-SOP5530kDa,PEG-SOP5540kDa,PEG-SOP5540kDa branched was performed in Lewis rat,and the serum concentration was detected to obtain the pharmacokinetic parameters of different molecular structures.Results：The test results showed that the binding efficiency between the antibody and target molecules was decreased by the PEGylated.The serum of rat and difference of polyethylene glycol molecular structures also affected the test result.By increasing the concentration of coating antibodies,target molecules were more efficiently captured.By replacing the dilution buffer of serum samples from PBS to imidazole buffer,and keeping the dilution rates of the standard curve and sample consistently,the interference of serum matrix was overcome.By maintaining the consistent polyethylene glycol molecules structure of the standard curve and sample,the interference caused by the difference of the polyethylene glycol molecules structures was overcome.The results of method validation showed that the method is specific,with a recovery rate of（100±15.2）%;the

关 键 词：聚乙二醇化 重组蛋白 聚醚高分子化合物 浓度测定 药代动力学 方法学验证 酶联免疫吸附实验(ELISA) 

分 类 号：R917[医药卫生—药物分析学]