α-Gal抗原定量检测试剂盒的研发

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&#215;10¹²个/每反应，检测回收率80%~120%；相关系数R²>0.98；6批次的板内、板间检测的相对标准偏差RSD<5%；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mrna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）×10^{14个&#183;mg-1，产品Gal抗原含量为（4.16&#177;2.56）&#215;1012 个&#183;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。}

objective:To optimize the α-Gal antigen detection method and to improve the stability of related reagents,to develop α-Gal antigen quantitative detection Kit,and evaluate the stability,reliability and applicability of the Kit. Methods:Based on the α-Gal antigen detection method,inhibitive enzyme linked immunosorbent assay(ELISA)in the medical device industry standard(YY/T 1561-2017,Tissue engineering medical device products-remnant α-Gal antigen determination in scaffold materials utilizing animal tissues and their derivatives),the following were further optimized:the reaction conditions of primary antibody,enzyme-labeled second antibody and solid-phase antigen-coating by evaluation of the recovery ratio,correlation coefficient R²,maximum absorbance value and maximum binding inhibition rate of primary antibody.And by combining with pre-prepared antigen-coated plate and ready-to-use reagents a Kit was developed.The stability of the Kit was evaluated by investigating the Gal antigen content of Gal-BSA reference material,the detection recovery rate of the Kit,the R² value of the standard curve,the maximum reaction absorbance(A)and the maximum binding inhibition rate.The Gal antigen of several samples was detected by optimized Kit,and the applicability of the Kit was investigated.Results:The minimum detection limit of Gal antigen of the optimizedk it was 5.64&#215;10¹² per reaction,the recovery rate of detection was 80%-120%;the correlation coefficient R² was more than 0.98;the RSD value of internal and among plate of 6 batch plates was less than 5%.There was no Gal antigen was detected in the negative reference material.The use of the Kit can accurately detect Gal antigen in organs of mice, and the results showed that the trend of Gal antigen expression to be detected in the main organs of mice was very consistent with the trend of mRNA expression which regulated the Gal antigen expression,indirectly evidenced the accuracy and reliability of the Kit.The results of Gal antigen content detected by the Kit in the raw material and the product of biological Dural mater patch were(8.34&#177;1.17)&#215; 10¹⁴&#183;mg^-1 wet tissues and(4.16&#177;2.56)&#215;10¹²&#183;mg^-1 wet tissues,and the antigen clearance rate was 99.5%.Conclusion:This Kit has good sensitivity and specificity,and it is suitable for detecting the quantity of α-Gal antigen residue and the clearance of Gal antigen of animal source medical device/biological material according to the industry standard YY/T 1561-2017,and provides technical support for the implementation of the industry standard.