摘要
旨在建立检测血清大豆抗原蛋白抗体的间接ELISA方法。经琼脂糖凝胶层析纯化大豆抗原蛋白,以不同剂量皮下注射免疫小鼠,采用方阵滴定法确定最佳抗原包被浓度及血清稀释度,并对其他条件进行优化,最终建立检测血清大豆抗原蛋白抗体的间接ELISA方法,利用该方法检测小鼠免疫后血清抗体水平。通过方阵滴定法确定11S蛋白最佳包被浓度为5.0μg/mL,血清稀释倍数为1∶800;7S蛋白抗原最佳包被浓度为2.5μg/mL,血清稀释倍数为1∶1 600;两者的批内、批间系数均小于10%,重复性较好,通过ELISA法确定11S和7S蛋白的最佳免疫次数为2次,免疫剂量为1 000μg/kg。结果表明本试验初步建立大豆抗原蛋白抗体检测间接ELISA方法,具有很强的特异性、敏感性和重复性,可用于大豆抗原蛋白过敏反应的临床检测。
The study is aimed to develop an indirect enzyme-linked immunosorbent assay （ELISA） for detection of serum specific antibod- ies of soybean antigen protein. Soybean antigen proteins were purified by Agarose gel chromatography and used to inject Kunming mice with different doses. An indirect ELISA was developed with the purified antigen protein at different concentrations, and the optimal antigen con- centration and serum dilution were determined by phalanx titration. The other working conditions were also optimized. The serum antibody levels of immuned mice were detected by the established ELISA. The optimal coating concentration of 11S protein and serum dilution were 5.0 μg/mL and 1 ： 800, respectively. The optimal coating concentration of 7S protein and serum dilution were 2. 5 μg/mLand 1 ： 1600, respectively. Both intra- and inter-assay repeatability of ELISA were less than 10%. The optimal immune times and doses of 11S and 7S protein were 2 and 1000 μg/mL , respectively. The developed indirect ELISA had good specificity, sensitivity and reproducibility. This study provides a reliable method for the clinical detection of allergic reactions induced by soybean antigen protein.
出处
《畜牧与兽医》
北大核心
2013年第1期19-24,共6页
Animal Husbandry & Veterinary Medicine
基金
安徽省教育厅自然科学重点科研项目(KJ2009A036)
