| Soybean is rich in a lot of nutrients. However, it also has become one of the main sources of dietary allergens for both humans and animals because of its antinutritional factors (ANFs). Glycinin and β-conglycinin are two major antigenic ANFs with good thermal stability. A reliable detection technology is urgently needed for precise detection of glycinin which will help make great progress in anti-nutrition mechanism research. This study performs a sandwich enzyme-linked immunosorbent assay (ELISA), which is applied for detection of trace amounts of glycinin in different kinds of soybean, as well as other soybean products. The research is divided into three parts. Experiment one:mouse anti-glycinin monoclonal antibody (Mab6F2) was prepared as a coating antibody, while the rabbit anti-glycinin polyclonal antibody (Pab) was applied as a secondary antibody. Experiment two:establish the sandwich enzyme-linked ELISA with the two antibodies above. The assay showed high specificity for glycinin with minimum cross-reactions with other soy proteins. By using the developed assay, the practical working range of the determination of glycinin was3～200ng/mL, and the limit of determination (LOD) was1.63ng/mL. The regaining of glycinin in spiked soybean samples were between93.8%and103.3%with relative standard deviation less than8.3%. Experiment three:the developed assay was used in analyzing469soybean seed samples from different sources, as well as five soybean products with different processing technique treatments. The data showed that the concentration of glycinin decreased significantly after certain kinds of processing techniques, particularly after fermentation of soybean meal, the concentration of glycinin almost dropped to zero. The assay provides a specific and sensitive method for the screening of glycinin and allows for further investigation into hypersensitive mechanisms to soybean proteins and development of soybean processing techniques to reduce their antinutritional effects. |
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