破伤风抗体体外结合ELISA检测方法的建立及初步验证

Establishment and validation of tetanus antibody in vitro binding enzyme-linked immunosorbent assay

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 and validate a tetanus antibody in vitro binding enzyme-linked immunosorbent assay.Methods Sera of mice immunized with tetanus vaccine were bound with bound toxoid（TT） in vitro, and transferred to the coated microtitration plate, onto which the second antibody, enzyme-labeled antibody and substrate were added to develop a tetanus antibody in vitro binding enzyme-linked immunosorbent assay. The developed method was validated for specificity, linear range, detection limit, reproducibility and accuracy. Results The linear range of standard curve of the developed method was 0. 016 ~ 1 U/ml, with a correlation coefficient of 0. 999 8, while the limit of detection was0. 009 U/ml. No cross reactions with diphtheria and pertussis antibodies were observed. The coefficient of variation（CVs） of high and low concentration quality control sera were 4. 467% and 5. 419% respectively, while the relative deviations were-8. 03% and-14% respectively. Conclusion The developed tetanus antibody in vitro binding enzymelinked immunosorbent assay showed high specificity, reproducibility and accuracy, which laid the foundation for establishing new potency tseting method of tetanus vaccines.