α-Gal抗原定量检测试剂盒的研发

发布时间：2018-04-29 06:27

本文选题：动物源医疗器械 + 生物材料　；参考：《药物分析杂志》2017年10期

【摘要】：目的:通过优化α-Gal抗原检测的方法及改善相关试剂的稳定性,研发α-Gal抗原定量检测试剂盒,并评价该试剂盒的稳定性、可靠性和适用性。方法:以医疗器械行业标准(YY/T 1561—2017组织工程医疗器械产品动物源性支架材料残留α-Gal抗原检测)中的α-Gal抗原检测酶联免疫抑制方法为基础,通过评价回收率、相关系数R2、最大吸收值和一抗的最高结合抑制率,对一抗、酶标二抗和固相抗原包被反应条件进行优化。预先制备好抗原包被板以及即用型试剂,制备成套试剂盒。通过考察Gal-BSA标准品的Gal抗原含量,评价试剂盒的检测回收率、标准曲线的R2值、最高反应的吸收度(A)和最高结合抑制率,考察其稳定性。使用优化好的试剂盒检测几种样品的Gal抗原,考察试剂盒的适用性。结果:经过优化后的试剂盒Gal抗原最低检测限为5.64×1012个/每反应,检测回收率80%~120%;相关系数R20.98;6批次的板内、板间检测的相对标准偏差RSD5%;阴性参考品无Gal抗原检出。使用该试剂盒能够准确检测小鼠脏器Gal抗原含量,其结果显示小鼠各脏器Gal抗原表达趋势与Gal抗原调控基因的m RNA表达趋势完全一致,间接地佐证了该试剂盒的准确性和可信性。检测生物型硬脑膜补片原材料Gal抗原含量为(8.34±1.17)×1014个·mg-1,产品Gal抗原含量为(4.16±2.56)×1012个·mg-1,计算得抗原清除率为99.5%。结论:该试剂盒具有较好的灵敏度,特异性,适用于依照行业标准YY/T 1561—2017进行动物源医疗器械/生物材料α-Gal抗原残留量的定量和Gal抗原清除率检测,为行业标准的实施提供技术保障。
[Abstract]:Objective: to develop and evaluate the stability, reliability and applicability of 伪 -Gal antigen quantitative detection kit by optimizing the method of 伪 -Gal antigen detection and improving the stability of related reagent. Methods: based on the detection of 伪 -Gal antigen in the tissue engineering medical device product animal source scaffold material, the method of Elisa was used to evaluate the recovery rate of 伪 -Gal antigen in the medical device industry standard (YY / T 1561-2017). The correlation coefficient R2, the maximum absorption value and the highest binding inhibition rate of the first antibody were optimized. The reaction conditions of the first antibody, the enzyme labeled second antibody and the solid phase antigen coating were optimized. A complete kit was prepared by preparing the antigen coating plate and the self-use reagent. The stability of Gal-BSA was investigated by investigating the Gal antigen content of the standard Gal-BSA, the detection recovery of the kit, the R2 value of the standard curve, the highest absorptivity of the reaction and the highest binding inhibition rate. The Gal antigens of several samples were detected by the optimized kit, and the applicability of the kit was investigated. Results: the minimum detection limit of Gal antigen of the optimized kit was 5.64 脳 1012 per reaction, the detection recovery was 80 ~ 120, the correlation coefficient was R20.98 ~ 6 batches, the relative standard deviation of intraplate detection was RSD _ 5 and the negative reference was not detected by Gal antigen. The results showed that the expression trend of Gal antigen in mouse viscera was consistent with that of m RNA gene regulated by Gal antigen. The accuracy and credibility of the kit are proved indirectly. The content of Gal antigen in raw material of biological dura mater patch was 8.34 卤1.17 脳 10 14 mg -1, and the content of Gal antigen in product was 4.16 卤2.56 脳 1012 mg -1. The antigen clearance rate was 99.5%. Conclusion: the kit has good sensitivity and specificity. It is suitable for the quantitative determination of 伪 -Gal antigen residues and the clearance rate of Gal antigen in animal medical devices / biomaterials according to industry standard YY/T 1561-2017. To provide technical support for the implementation of industry standards.
【作者单位】：北京三药科技开发公司;中国食品药品检定研究院;北京万泰生物药业股份有限公司;
【基金】：再生型医用植入器械国家工程实验室PI项目(课题编号:2012NELRMD002) 科技部生物医用材料研发与组织器官修复替代重点专项(2016YFC1103200,2016YFC1103203)
【分类号】：R392-33

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