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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~(-1),t_(1/2)分别为15.6、22.3、30.6 h。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·m L~(-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。
OBJECTIVE: To establish an ELISA method for the determination of the concentration of pegylated recombinant protein (PEG-SOP55) in rat serum and to study the pharmacokinetics of the protein. Methods: The polyclonal antibody was coated with biotin, labeled with biotin, amplified with biotin-streptavidin-horseradish peroxidase (HRP) and amplified by double enzyme-linked immunosorbent assay Detection. The basic experimental conditions of this method were established by optimizing the working concentration of coating antibody and detection antibody, interference experiment of serum matrix and comparison experiment of polyethylene glycol molecules with different structures. And reference to relevant laws and regulations, its method specificity, accuracy, precision, durability, detection limits and sample stability verification. Lewis rats were intraperitoneally administered with PEG-SOP55_ (30 kDa), PEG-SOP55_ (40 kDa) and PEG-SOP55_ (40 kDa branched), respectively, and serum concentrations were measured to obtain the protein modified with polyethylene glycol molecules of different molecular structures After the pharmacokinetic parameters. Results: The experiment confirmed that polyethylene glycol modified the binding efficiency of the detection antibody and the target molecule, and the changes of the molecular structure of the serum and polyethylene glycol in the rat interfere with the detection. By increasing the coating antibody concentration, better capture efficiency is achieved for the target molecule. By replacing the serum sample dilutions from the PBS to the imidazole buffer and keeping the standard curve in line with the serum dilution of the test sample, interference from the serum matrix is overcome. By keeping the standard curve consistent with the polyethylene glycol molecular structure of the sample to be tested, the interference caused by the different molecular structure of the polyethylene glycol is overcome. The method validation showed that the method was of good specificity and the recovery rate was (100 ± 15.2)%. The intra-laboratory precision and inter-laboratory precision verification data RSD did not exceed 20%, and different operators had no significant effect on the test results. The standard curve Good linearity, the highest and lowest concentration of the standard curve can be accurately determined. Rat serum samples were repeatedly frozen and thawed three times at room temperature for 4 h the stability of the meet the requirements to meet the detection conditions of the method. The pharmacokinetic parameters of PEG-SOP55_ (30kDa), PEG-SOP55_ (40kDa) and PEG-SOP55_ (40kDa branched) were determined by concentration determination of Lewis rat serum samples. The maximum values of C_ (max) were 31.9, 54.4 and 67.7 mg · m L -1, and the AUC_ (0-∞) were 823.0, 1 978.0 and 3 502.6 mg · h · m L -1, respectively, (1/2) were 15.6,22.3,30.6 h respectively. Conclusion: The ELISA method for detecting the concentration of PEG-SOP55 in rat serum has been successfully established. The method validation shows that the method has good specificity, accuracy, precision, linearity and durability. The determination range is 5 ~ 80 ng · m L -1. The stability of the rat serum sample meets the detection requirements of the method. The method can be used as a detection method for the PEG-SOP55 pharmacokinetic study, and provide a reference for the serum concentration detection of similar products. Preliminary pharmacokinetic studies have shown that with the polyethylene glycol molecules on the shielding effect of protein increased, can significantly extend its half-life and improve the drug exposure in the body.