小鼠血清中杀O139霍乱弧菌抗体检测方法的初步建立

目的研究探索O139霍乱弧菌杀弧菌抗体的检测方法。方法用微孔板培养和琼脂平板克隆计数相结合的杀弧菌抗体检测方法,对实验菌株及稀释度、补体浓度等关键参数进行筛选;对50份小鼠免疫血清进行杀弧菌抗体滴度检测,并与O139群霍乱弧菌LPS Ig G抗体滴度进行相关分析;对该方法的特异性、线性和精密性进行了验证。结果筛选出最佳菌株为20100603菌株,最佳稀释度倍数为2 000倍,补体最佳稀释倍数为16倍。O139群霍乱弧菌小鼠免疫血清检测到较高的杀弧菌抗体滴度而PBS小鼠免疫血清未检测到杀弧菌抗体滴度。小鼠免疫血清杀弧菌抗体滴度与O139群霍乱弧菌LPS Ig G抗体滴度之间存在正相关关系。验证结果显示,在抑制剂浓度达到1.0~2.0 A600时,抑制率100%;线性回归方程为y=-1.093x+5.058,其相关系数为-0.999,P0.05;方法批内CV值为15.72%,批间CV值为23.47%。结论初步建立了O139霍乱弧菌杀弧菌抗体的检测方法,该方法具有较高的特异性、线性和精密度。

Preliminary establishment of a vibriocidal assay method for the detection of bactericidal antibodies to Vibrio cholerae O139

Objective To establish a vibriocidal assay method for the detection of bactericidal antibodies against Vibrio cholerae O139 . Methods A vibriocidal assay method coupling 96-well microtiter-plate format with automated colony counting on agar plate was used to detect serum vibriocidal titer. Tested bacteria strain, tested bacteria dilution ratio and concentration of complement was optimized , respectively. Vibriocidal titer of 50 mice sera immunized with Vibrio cholerae O139 bacteria or PBS were detected with above method. Specificity,linearity and precision of method was validated. Re- lusts Optimum bacteria strain was 20160603, dilution ratio was 1 ： 2 000, and the best concentration of complement was 1 ： 16. Vibriocidal titers of mice sera immunized with Vibrio cholerae O139 were high, while vibriocidal titers of mice sera immunized with PBS were low. Vibriocidal titer was 100% inhibited when concentration of inhibitor was 1.0-2.0 A600, lin- ear regression equation was y=-l. 093X＋5. 058, correlation coefficient（r） = -0. 999, P〈0.05, the within-run and be- tween-run CV were 15.72% and 23.47% , respectively. Conclusion A vibriocidal assay method for the detection of bac- tericidal antibodies to Vibrio cholerae O139 has been preliminary established, which was specific with good linearity and precision.