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刊物信息
期刊名称:药物分析杂志
主管单位:中国科学技术协会
主办单位:中国药学会
承办:中国食品药品检定研究院
主编:金少鸿
编辑部主任:粟晓黎
地址:北京天坛西里2号
邮政编码:100050
电话:010-67012819,67058427
电子邮箱:ywfx@nicpbp.org.cn
国际标准刊号:ISSN 0254-1793
国内统一刊号:CN 11-2224/R
邮发代号:2-237

 

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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

分类号:
出版年·卷·期(页码):2016,36 (10):0-0
DOI: 10.16155/j.0254-1793.2017.01.01
-----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-SOP5530 kDa、PEG-SOP5540 kDa、PEG-SOP5540 kDa 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-SOP5530 kDa、PEG-SOP5540 kDa、PEG-SOP5540 kDa branched的药代动力学参数。其Cmax分别为31.9、54.4、67.7 mg·mL-1,AUC0-∞分别为823.0、1 978.0、3 502.6 mg·h·mL-1t1/2分别为15.6、22.3、30.6h。结论:成功建立检测PEG-SOP55在大鼠血清中浓度的ELISA方法。通过方法验证表明:该方法专属性、准确度、精密度、线性以及耐用性良好,测定范围为5~80 ng·mL-1。大鼠血清样品稳定性满足该方法检测要求,该方法可作为PEG-SOP55药代动力学研究的检测方法,也为类似制品的血清浓度检测提供借鉴。初步药代动力学研究表明,随着聚乙二醇分子对蛋白的屏蔽作用增强,能够显著延长其半衰期,提高药物在体内的暴露量。

-----英文摘要:---------------------------------------------------------------------------------------

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 PEGSOP5530 kDa, PEG-SOP5540 kDa, PEG-SOP5540 kDa 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 relative standard deviations (RSD)of the data of repeatability and intermediate precision were all below 20%; different operators had no significant effect on the test results. Linearity of the standard curve is good, and the minimum and maximum concentrations of the standard curve can be determined accurately. By freezing and thawing of the rat serum samples 3 times, and keeping the rat in room temperature 4h, the sample remained stable. The pharmacokinetics parameters of PEG-SOP5530 kDa, PEG-SOP5540 kDa,PEGSOP5540 kDa branched were obtained by measuring the concentration of the serum samples of Lewis rats. The Cmax were 31.9 mg·mL-1,54.4 mg·mL-1,67.7 mg·mL-1,respectively;the AUC0-∞ were 823.0 mg·h·mL-1,1 978.0 mg·h·mL-1,3 502.6 mg·h·mL-1;the t1/2 were 15.6 h,22.3 h,and 30.6 h,respectively. Conclusion: An ELISA method was successfully established to detect the content of PEG-SOP55 in serum of rats. The specificity, accuracy, precision,linearity,and robustness of this method were validated. The detection range of this method is 5-80 ng·mL-1. The stability of the serum sample meets the request of the detection method. This method can be used to detect the serum samples of PEG-SOP55 pharmacokinetics, which can provide a basis for similar PEGylated protein. Preliminary pharmacokinetic study revealed that the increase of the protein's shielding effect which caused by the polyethylene glycol molecule could significantly prolong its half-life and increase the exposure of the drug in vivo.

-----参考文献:---------------------------------------------------------------------------------------
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