State Key Laboratory of Animal Nutrition, China Agricultural University, Beijing 100193, China.
Anal Chim Acta. 2012 Jul 13;734:62-8. doi: 10.1016/j.aca.2012.05.009. Epub 2012 May 15.
Soybean protein has long been recognized as a source of dietary allergens for humans and animals with β-conglycinin being the major allergen. This paper presents a sandwich enzyme-linked immunosorbent assay (ELISA) that allows for the detection of trace amount of β-conglycinin in soybean and soybean products. In the sandwich ELISA, mouse anti-β-conglycinin monoclonal antibody (Mab 5C5) was used as coating antibody, and rabbit anti-β-conglycinin polyclonal antibody (Pab) was used as secondary antibody. The assay showed high specificity for β-conglycinin with minimum cross-reactions with other soy proteins. The practical working range for the determination of β-conglycinin using the developed assay was 3-100ngmL(-1) and the limit of determination (LOD) was 1.63 ng mL(-1). The recoveries of β-conglycinin in spiked soybean samples were between 88.1% and 106.6% with relative standard deviation less than 8.9% (intra-day) and 13.1% (inter-day). The developed method was used to analyze 469 soybean seed samples from different sources as well as five soybean products treated with different processing techniques. The data showed that the concentration of β-conglycinin decreased significantly after processing, especially for soybean protein isolation, where the concentration of β-conglycinin dropped to nearly zero. The assay provides a specific and sensitive method for the screening of β-conglycinin and allows for further investigation into hypersensitive mechanisms of soybean proteins and development of soybean processing techniques to reduce their negative effects.
大豆蛋白一直被认为是人类和动物的膳食过敏原，其中β-伴大豆球蛋白是主要过敏原。本文提出了一种夹心酶联免疫吸附测定（ELISA）方法，可用于检测大豆和大豆制品中痕量的β-伴大豆球蛋白。在夹心 ELISA 中，使用小鼠抗β-伴大豆球蛋白单克隆抗体（Mab 5C5）作为包被抗体，兔抗β-伴大豆球蛋白多克隆抗体（Pab）作为二级抗体。该测定法对β-伴大豆球蛋白具有高度特异性，与其他大豆蛋白的交叉反应最小。使用所开发的测定法测定β-伴大豆球蛋白的实际工作范围为 3-100ngmL(-1)，测定限（LOD）为 1.63ngmL(-1)。在添加的大豆样品中β-伴大豆球蛋白的回收率在 88.1%至 106.6%之间，日内相对标准偏差小于 8.9%，日间相对标准偏差小于 13.1%。该方法用于分析来自不同来源的 469 个大豆种子样品以及经过不同加工技术处理的五种大豆产品。数据表明，加工后β-伴大豆球蛋白的浓度显著降低，尤其是在大豆蛋白分离过程中，β-伴大豆球蛋白的浓度几乎降至零。该测定法为β-伴大豆球蛋白的筛选提供了一种特异而灵敏的方法，并允许进一步研究大豆蛋白的过敏机制和开发降低其负面影响的大豆加工技术。
