摘要
Aim To develop a sensitive competitive ELBA for the determination of biotinin transformed yeast culture media. Methods The ELBA plate was firstly coated with Mycoplasmahyopneumoniae, and then successively incubated with rabbit anti-Mycoplasma hyopneumoniae serum andgoat 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 standardcalibration curve for biotin analysis was constructed in the range of 50 - 2000 ng·L^(-1). ResultsThe detection limit for biotin was found to be 83 ng·L^(-1), which was about 1000 times lower thanthe lowest determination concentration in the reported ELISA for biotin analysis. The relativestandard deviations for the spiked samples at biotin concentrations of 200 ng·L^(-1), 500ng·L^(-1), and 1000 ng·L^(-1) were 24.87%, 6.15% , and 7.86% , respectively, with the averagerecovery of 101.13% . The wild yeast and its sixty-three transformed yeast culture media wereapplied to the developed ELBA for the determination of biotin. It was found that the biotinconcentrations in more than 85% of the tested samples were enhanced with different increase factorsafter transformation. Conclusion Utilization of Mycoplasma hyopneumoniae as the coating proteinimproves the precision and accuracy of the ELBA assay, which might be used for the biotin assay inother media.
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 ami-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 standard calibration curve for biotin analysis was constructed in the range of 50-2000ng·L^-1. Results The detection limit for biotin was found to be 83 ng·L^-1 , which waa about 1000 times lower than the lowest determination concenlration in the reported ELISA for biotin analysis. The relative standard deviations for the spiked samples at biotin concerarations 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 enhanced with different increase factors after transformation. Conclusion Utilization of Mycoplasma hyopnetunoniae as the coating protein improves the precision and accmacy oftbe ELISA assay, which might be used for the biotin assay in other media.
