酶联反应板包被链霉亲合素的优化方法

Optimal conditions of coating streptavidin on microtitration plates

目的对链霉亲合素(streptavidin,SA)预包被酶联反应板的优化方法进行研究,比较干法、湿法包被的优越性。方法通过对包被缓冲液、SA浓度、17种市售反应板的比较试验,得出SA包被的较优条件。结果选择洁特高亲和力板,采用碳酸缓冲液干法包被,37℃温育16 h至全干,达最佳包被效果,此时SA浓度为2μg/ml。干法包被的孔间差2.95%,批间差7.37%,37℃保存7 d结合生物素的效果无显著改变。生物素结合量为每孔1 ng,是国产SA预包被反应板的5倍。结论用干法预包被SA优于传统的湿法包被,效果等同甚至超过现有进口SA预包被板。

Objective This study aimed to determine optimal coating conditions for streptavidin （SA） immobilization onto microtitration plate by means of comparing the advantages of dry-coating and wet-coating. Methods Various concentrations of SA in different coating buffers and 17 different brands of commercially available microtitration plates were tested by dry-coating and wet-coating methods respectively. The coating stabilities and coating-related variations were analyzed. Results The optimal dry-coating protocol was that Jiete high-binding plate was coated with 100 μl of 2 μg/ml SA in 0.05 mol/L carbonate buffer （pH 9.6） at 37 ℃ for 16 h until the buffer was evaporated. The coefficients of well and batch variation were 2.95% and 7.37% respectively. The biotin-binding value was 1 ng per weft, which was as 5 times as that in domestic microtitration plates. Moreover, the coating efficiency kept stable after incubated at 37 ℃ for 7 days. Conclusions The SA dry-coating method developed in the study was proved to be more efficient than the conventional wet-coating. The quality of the SA coated microtitration plates was as good as or even better than the imported plates.