霍乱疫苗中残余霍乱毒素检测方法的建立及验证

The establishment and validation of method for detecting residual cholera toxin in cholera vaccine

采用间接酶联免疫法,即用神经节苷脂包被,加入待检样品,再加入兔抗霍乱毒素B亚单位抗体,用标准样品的吸光值(A值)对标准样品的浓度绘制4-参数拟合曲线,根据标准曲线计算出待测样品中的CT浓度。结果显示,在浓度范围(0.6～16)ng/ml之间,CT标准浓度和检测浓度成线性关系,r2=0.9986。精确度在浓度范围(0.6～16)ng/ml,CT的平均回收率在96.24%～114.44%之间。精密度:批内变异CV%≤12.98%,批间变异CV%≤18.48%。特异性CT浓度在10ng/ml时,平均回收率为102.6%;CT浓度在5ng/ml时,平均回收率为111.17%;CT浓度在2.5ng/ml时,平均回收率为123.83%。实验表明该方法可检测霍乱疫苗原液中CT的含量。

To establish a method for detecting residual cholera toxin in cholera vaccine.By using a indirect enzyme-linked immunosorbent assay,briefly,the samples were added to microtiter plates previously coated with ganglioside GM1,then the rabbit anti-cholera toxin B subunit antibody was added.4-parameter fitting curve was plotted against the absorbance values(A)and the concentration of standard samples.CT concentrations in the tested samples were calculated according to the standard curve.The results showed that when the concentration is(0.6 - 16)ng/ml,CT standard concentration and the detected concentration has a linear relationship,r2=0.9986.Degree of accuracy: when the concentration is(0.6 - 16)ng/ml,the average recovery rate can reach 96.24% - 114.44%.Degree of precision: the CV of within group≤12.98%,the CV between groups≤18.48%.Specificity: when the concentration of CT is 10ng/ml,5ng/ml and 2.5ng/ml,the average recovery rate is 102.6%,111.17% and 123.83% separately.This method can be used to detect the CT concentration in cholera liquid vaccine.