抗营养因子大豆凝集素夹心ELISA检测方法的建立  

Establishment of a Sandwich ELISA for Detecting Soybean Agglutinin as an Anti-nutritional Factor

作　　者：胡骁飞[1] 杨继飞[1] 邢云瑞 庞杏豪 孙亚宁[1] 魏凤仙[2] HU Xiaofei;YANG Jifei;XING Yunrui;PANG Xinghao;SUN Yaning;WEI Fengxian(Key Laboratory for Animal Immunology,Henan Academy of Agricultural Sciences,Zhengzhou 450002,China;Institute of Husbandry,Henan Academy of Agricultural Sciences,Zhengzhou 450002,China)

机构地区：[1]河南省农业科学院动物免疫学重点实验室,郑州450002 [2]河南省农业科学院畜牧研究所,郑州450002

出　　处：《西北农业学报》2025年第1期133-139,共7页Acta Agriculturae Boreali-occidentalis Sinica

基　　金：国家重点研发计划(2018YFC1602903);河南省现代农业产业技术体系项目(HARS-SS-12-S)。

摘　　要：为建立灵敏、特异的大豆凝集素(soybean agglutinin,SBA)酶联免疫吸附(enzyme-linked immuno sorbent assay,ELISA)检测方法。试验以SBA鼠源单克隆抗体为捕获抗体,SBA兔源多克隆抗血清为检测抗体,羊抗兔酶标抗体为二抗,通过对抗体的工作浓度及反应时间优化,建立SBA的双抗体夹心ELISA检测方法,并用建立的方法检测大豆、大豆粕、膨化大豆粕及酶解大豆粕中SBA含量。结果表明:建立SBA双抗夹心ELISA检测方法的检测范围为39.06~1250 ng/mL,检出限为3.00 ng/mL,样品添加回收率为77.96%~116.15%,变异系数为6.11%~18.34%,与大豆中胰蛋白酶抑制因子BBI、KTI,大豆球蛋白glycinin及大豆伴球蛋白-conglycinin等其他抗营养因子均无交叉反应。大豆中SBA含量显著高于大豆粕、膨化大豆粕及酶解大豆粕(P<0.05);大豆粕中SBA含量显著高于膨化大豆粕及酶解大豆粕(P<0.05);膨化大豆粕显著高于酶解大豆粕中SBA含量(P<0.05)。综上,本研究建立了灵敏度高、特异性好的SBA双抗夹心ELISA检测方法,该方法可用于饲料中大豆凝集素的监控检测。To establish a sensitive and specific enzyme-linked immunosorbent assay(ELISA)for the detection of soybean agglutinin(SBA).Mouse-derived monoclonal antibody was used as the capturing antibody and rabbit-derived polyclonal antibody as the detecting antibody,with a sheep anti-rabbit enzymic conjugate antibody as the secondary antibody.This sandwich method for SBA detection was optimized in terms of antibody concentration and reaction time.This method was applied to detect SBA levels in differenet soybean products,including soybean,soybean meal,extruded soybean and enzyme hydrolyzed soybean meal.The results showed that the detection range of the developed sandwich ELISA was 39.06-1250 ng/mL,the detection limit was 3.00 ng/mL,the fortified recovery was 77.96%-116.15%,the coefficient of variation was 6.11%-18.34%.There was no cross-reaction with other anti-nutritional factors such as trypsin inhibitors BBI,KTI,glycinin andβ-conglycinin in soybean.The concentration of SBA in soybean was higher than that in soybean meal,extruded soybean meal and enzyme hydrolyzed soybean meal,respectively(P<0.05).SBA in soybean meal was higher than in extruded soybean meal and enzyme hydrolyzed soybean meal,respectively(P<0.05)and in extruded soybean meal was higher than in enzyme hydrolyzed soybean meal(P<0.05).These findings indicate that the established SBA sandwich ELISA detection method is both sensitive and specific and can be used for monitoring and detecting soybean agglutinin in feed.

关 键 词：大豆凝集素 抗营养因子 多克隆抗体 单克隆抗体 夹心ELISA 

分 类 号：S816.17[农业科学—饲料科学]