论文部分内容阅读
目的研制蛋白酶K单克隆抗体并构建定量检测蛋白酶K的双抗体夹心ELISA方法 ,用于生物制品中蛋白酶K的检测。方法用杂交瘤技术制备抗蛋白酶K的单抗,用棋盘法优化ELISA条件,建立定量检测蛋白酶K的双抗体夹心ELISA方法,分析其特异性、精密性和准确性。结果经免疫、融合、克隆、筛选到稳定分泌蛋白酶K单抗的杂交瘤细胞。获得1株阻断蛋白酶K活性的单抗5G6,以5G6为包被抗体、4D3为酶标抗体,构建蛋白酶K定量检测的ELISA方法。本方法线性相关系数R=0.99,线性范围为293.4~2 500 pg/ml;定量限度为293.4 pg/ml;变异系数为CV<15%;回收率介于89.3%~129.2%之间,37℃6 d的回收率>80%;与蛋白酶K以外的其他蛋白无交叉反应。结论获得阻断蛋白酶K活性的单抗,构建出灵敏、特异的蛋白酶K定量检测方法。
Objective To develop a monoclonal antibody against protein K and to construct a double antibody sandwich ELISA for the quantitative detection of proteinase K for the detection of proteinase K in biological products. Methods The monoclonal antibodies against proteinase K were prepared by hybridoma technique. The conditions of ELISA were optimized by the checkerboard method. The double antibody sandwich ELISA method was established for the quantitative detection of proteinase K. The specificity, precision and accuracy of the ELISA were analyzed. Results After immunization, fusion and cloning, hybridoma cells stably secreting proteinase K monoclonal antibody were screened out. Obtained a monoclonal antibody 5G6 that blocked the activity of protease K, 5G6 as coated antibody and 4D3 as enzyme-labeled antibody. The linear correlation coefficient of this method was 0.99, the linear range was 293.4-2 500 pg / ml, the limit of quantification was 293.4 pg / ml, the coefficient of variation was CV <15%, and the recoveries ranged from 89.3% to 129.2% The recovery rate of 6 days was> 80%. There was no cross reaction with other protein except proteinase K. Conclusion A monoclonal antibody that blocks the activity of proteinase K was obtained and a sensitive and specific method for the quantitative detection of proteinase K was constructed.