基因转殖酵母菌培养液中生物素的高灵敏ELISA竞争测定法

基因转殖酵母菌培养液中生物素的高灵敏ELISA竞争测定法

杨红^*

四川大学华西药学院, 成都 610041

收稿日期:2003-02-06 修回日期:2003-11-10 出版日期:2003-12-15 发布日期:2003-12-15
通讯作者: 杨红*

A Sensitive Competitive ELISA for Determination of Biotin in Transformed Yeast Culture Media

YANG Hong^*

West China School of Pharmacy, Sichuan University, Chengdu 610041, China

Received:2003-02-06 Revised:2003-11-10 Online:2003-12-15 Published:2003-12-15
Contact: YANG Hong*

摘要/Abstract

摘要： 目的发展一种测定基因转殖酵母菌培养液中生物素的灵敏ELISA竞争测定法. 方法酶标板先用猪肺炎支原体包被, 再依次用兔抗猪肺炎支原体抗血浆、羊抗兔IgG-生物素温育形成固态生物素, 同溶液中的生物素(标准液或试样)竞争有限量的链霉亲和素-HRP.建立了在50-2000 ng·L^-1范围内生物素的标准曲线. 结果检测限为83 ng·L^-1, 比文献报告的ELISA测生物素的最低测定浓度小1000倍.测定生物素浓度为200、500、1000 ng·L^-1的用空白培养液稀释的标准生物素试样的相对标准偏差分别是24.87%、6.15%、7.86%, 平均回收率为101.13%.在用本法测定野生酵母菌及其63个基因转殖酵母菌培养液中的生物素时, 发现85%以上的培养液试样中生物素浓度均得到不同程度扩增. 结论用猪肺炎支原体作包被蛋白, 提高了ELISA结果的精确度和准确度, 可供测定其它介质中生物素的参考.

关键词: 亲和分析, 生物素测定, 链霉亲和素, 酶联免疫吸附测定法, 包被, 基因转殖酵母菌, 猪肺炎支原体

Abstract: Aim To develop a sensitive competitive ELISA for the determination of biotin in transformed yeast culture media. Methods The ELISA plate was firstly coated with Mycoplasma hyopneumoniae, and then successively incubated with rabbit anti-Mycoplasma hyopneumoniae serum and goat anti-rabbit IgG-biotin to form the solid biotin, which competed with the biotin in the solution (standard or sample) for the limited streptavidin-horse radish peroxidase conjugate. The stan-dard calibration curve for biotin analysis was constructed in the range of 50-2000 ng·L^-1. Results The detection limit for biotin was found to be 83 ng·L^-1, which was about 1000 times lower than the lowest determination concentration in the re-ported ELISA for biotin analysis. The relative standard deviations for the spiked samples at biotin concentrations of 200 ng·L^-1, 500 ng·L^-1, and 1000 ng·L^-1 were 24.87%, 6.15%, and 7.86%, respectively, with the average recovery of 101.13%. The wild yeast and its sixty-three transformed yeast culture media were applied to the developed ELISA for the determination of biotin. It was found that the biotin concentrations in more than 85 % of the tested samples were enhancedwith different increase factors after transformation. Conclusion Utilization of Mycoplasma hyopneumoniae as the coating protein improves the precision and accuracy of the ELISA assay, which might be used for the biotin assay in other media.

Key words: affinity assay, affinity assay, biotin analysis, biotin analysis, streptavidin, streptavidin, ELISA, ELISA, coating, coating, transformed yeast culture media, transformed yeast culture media, Mycoplasma hyopneumoniae, Mycoplasma hyopneumoniae

中图分类号:

R927.3

Supporting:

杨红^*. 基因转殖酵母菌培养液中生物素的高灵敏ELISA竞争测定法[J]. .

YANG Hong^*. A Sensitive Competitive ELISA for Determination of Biotin in Transformed Yeast Culture Media[J]. .

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