Devolopment Of Enzyme-Linked Immunassay For The Determination Of Mung Bean Allergen

It has not been reported that mung bean allergen were detected at home and abroad, but the allergen has the potential hazard to sensitive people. Therefore, to eatablish a simple and rapid detection method has important practical significance. In this paper, the sandwich enzyme-linked immunosorbent assay (ELISA) for the determination of mung bean allergen has been developed.Extracted mung bean protein, SDS-PAGE were carried out to assess the molecular weight of the mung bean protein, major bands were at50,38,34and25kDa. Protein concentration was measured by Brandford assay. Allergy reactions in BALB/C mice caused by mung bean protein. BALB/c mice were immunized by subcutaneous injection with Freund’s adjuvant. And the whole blood was collected at14days after the last booster. The titer of mice serum was1:64000. New Zealand White rabbits were immunized four times, the titer of rabbits serum was1:32000. Immunoblotting were carried out to assess the affinity of antibody. The mice and rabbits sera bind specifically to two major mung bean protein bands at about50and25kDa. The mung bean sandwich ELISA is highly specific for the detection of mung bean proteins and in the presence of nine kinds of other foods.Then through the optimization of the immunoassay conditions, the established sandwich ELISA was sensitive and rapid. Determined capture antibody coating quantity was5μg/well and skim milk was selected as blocking solution.0.01mol/L PBS (pH=7.4) was chose as running buffer of the assay. The LOD of the standard curve was4.99ng/mL Recoveries were between80.35%-88.41%. The intra-assay and inter-assay were1.58%-3.78%and3.14%-6.41%separately.The stability of food allergens on thermal processing was analyzed by sandwich ELISA. In the dry heat processing conditions, with increasing temperature, the mung bean protein has been caused more changes than mung bean powder. In the moist heat processing conditions, the antigens stability of mung bean protein had little change, while the antigens stability of protein solution had a significant decline.