用ELISA法测定基因转殖酵母菌发酵液中生物素

目的:采用一种简便、高灵敏度、高准确度的ELISA方法,对基因转殖可制造生物素的酵母菌发酵液中生物素含量的直接定量。方法:采用猪肺炎霉浆菌包被酶标板,再依次结合兔抗猪肺炎霉浆菌血清及生物素化羊抗兔IgG,形成生物素的固定相,用竞争抑制法测定生物素含量,各步实验条件均经优化。结果:测定的线性范围为 50～2 000ng/L,灵敏度I C_(50)=410ng/L,检测限为83 ng/L,线性相关系数r=-0.990;样品液1:1000稀释后,可消除基质效应的影响直接测定,加标平均回收率为101.13％。结论:用本文ELISA法测定基因转殖酵母菌发酵液中生物素的方法简便,结果可靠。

Study on an ELISA Method for Determination of Biotin in Yeast Cultural Media for Gene Transformation

Objective: To develop an ELISA method of simple, high sensitivity and accuracy for direct quantitation of biotin in the yeast cultural media for gene transformation. Method: Mycoplasm hyopneumoniae was used to coat the microtiter plate and its antibody in rabbit and goat anti-rabbit IgG incorporated biotin were linked to the plate successively to form a fixed phase of biotin which, after optimization of the experimental conditions, was applied in the followed competition inhibition reaction to determine biotin. Results: The linear range of determination was 50-2 000 ng. L-1 with a sensitivity of IC 50 = 410ng.L-1, a determination limit of 83 ng. L-1, and the correlation coefficient of r = - 0.990. After sample solution 1:1 000 was diluted, the cultural media was directly used for ELISA,and the mean recovery rate was 101.13% .The matrix effect was proved to be already eliminated.Conclusion:The developed method is simple and reliable for direct determination of biotin in the yeast cultural media for gene transformation.