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目的建立破伤风抗体体外结合ELISA检测方法,并进行初步验证。方法将破伤风疫苗免疫后获得的小鼠血清与破伤风类毒素(tetanus toxoid,TT)在体外进行结合,将结合后的血清转移至已包被的酶标板中,分别加入二抗、酶标抗体和底物等进行检测,建立破伤风抗体体外结合双抗夹心ELISA法。对建立的方法进行特异性、线性范围、检测限、重复性和准确性初步验证。结果建立的破伤风抗体体外结合双抗夹心ELISA法的标准曲线在浓度为0.016~1 U/ml之间时,可获得最佳线性,相关系数R=0.999 8;检测限为0.009 U/ml;与白喉-百日咳抗体无交叉反应;重复性验证中高浓度和低浓度质控血清的变异系数(CV)分别为4.467%和5.419%;准确性验证中高浓度和低浓度质控血清的相对偏差分别为-8.03%和-14%。结论建立的破伤风抗体体外结合ELISA检测方法具有良好的特异性、重复性和准确性,为破伤风疫苗效价检测新方法的建立奠定了基础。
Objective To establish a method for the detection of tetanus antibody in vitro by ELISA. Methods The serum from mice immunized with tetanus vaccine was combined with tetanus toxoid (TT) in vitro and the bound sera were transferred to the coated ELISA plates. Secondary antibodies and enzymes Standard antibodies and substrates were detected, the establishment of tetanus antibody in vitro binding double antibody sandwich ELISA method. The established method was validated by specificity, linear range, detection limit, repeatability and accuracy. Results The established standard curve of tetanus antibody in vitro with double antibody sandwich ELISA showed the best linearity at the concentration of 0.016-1 U / ml with a correlation coefficient of R = 0.999 8 and a detection limit of 0.009 U / ml. And the diphtheria-pertussis antibody did not cross-react. The coefficient of variation (CV) of the high-quality and low-quality control sera in repeated validation were 4.467% and 5.419%, respectively. The relative deviation of the high-quality and low- -8.03% and -14%. Conclusion The established tetanus antibody in vitro binding ELISA assay has good specificity, repeatability and accuracy, which laid the foundation for the establishment of a new method for the detection of tetanus vaccine titer.