Preparation Of Monoclonal Antibody Against Soybean Agglutinin And The Establishment Of Function ELISA

Soybean agglutinin (SBA), a protein present in raw soybean meals, can bind to and be extensively endocytosed by intestinal epithelial cells, being nutritionally toxic for most animals.As an anti-nutritional factors in animal feeds, food and other bean products could agglutinate red blood cells,effect digestion and absorption of nutrients, inhibit the growth of animals and cause allergic reactions,induce a series of anti-nutritional effects in animals and humans, serious impact on animal production and human health, therefore, detect the content of SBA accurately is important. There are some methods to detect the SBA but ench of method has its disadvantages. In this study, through the preparation of anti-SBA monoclonal antibodies and the specific biological activity of agglutination established the fast, accurate and stable functional ELISA method for detection of SBA.In this study,8weeks old BALB/c mice were immunised with the purified SBA, Established indirect enzyme-linked immunosorbent assay after the third immunization.The optimal working concentration of coating antigen is8μg/ml and the antiserum is1:8000.The ELISA was used to detect the antibody titer of antiserum and cell culture supernatantAfter strengthening immunization the titer of immune serum were6.4×104. The efficiency of fusional cells with50%polyethylene glycol was80%, and the rate of positive clone was31%. Two hybridoma cell lines producting anti-SBA monoclona antibodies were gained by the limited dilution method and hybridoma technique after4times subcloning named A7and F12.The antibody titers of cell culture supernatant was1:2x103and1:3×103and ascites were1:1×106.The affinity constants of A7、F12monoclonal antibody were4.88×107mol/L and7.1×107mol/L, and both of the subtypes of monoclonal antibody are IgG2b. The molecular weight of F12monoclonal antibodies was189.6KDa, the lambda chain was24.1KDa, gamma chain was70.7KDa, two monoclonal antibodies showed high specificity with SBA.To establish the function ELISA, red blood cell ghosts were coated onto microtitre plates, any SBA which retains it’s aggregate activity would bound to carbohydrates on the cell ghost surfaces, then the bound SBA was detected with anti-SBA McAb, and the bound McAb detected with rabbit anti-mouse IgG conjugated to horseradish peroxidase. In the function ELISA,the optimum working concentration of red blood cell ghosts was8μg/mL and the dilution of McAb was1:2000. The levels of SBA were measured by the standard curves constructed from serial dilutions of pure SBA. The function ELISA assay was confirmed to have good reproducibility and specificity.