Study On Immunological Rapid Detection Method Of Soybean Bowman-Birk Inhibitor

Soybean is an excellent resource of vegetable protein with rich and balanced essential amino acids.However,it also contains many kind of anti-nutrition factor and allergenic proteins,which not only will reduce the nutritional value but also harm the body,such as soybean trypsin inhibitor（STI）.Soybean trypsin inhibitor is one kind of important anti-nutritional factor and allergenic protein in soybean,which can not only induce the hypersensitivity reaction of the body,but also bind to trypsin and chymotrypsin of the body to inhibit their activities,and then lead to indigestion,eventually hinder growth and development.Therefore,it is especially necessary to conduct a study on the detection of STI.STI contains two main inhibitor,Kunitz trypsin inhibitor（KTI）and Bowman-Birk inhibitor（BBI）,wherein BBI is stable and is not easy to inactivate and remove.In this study,BBI was applied as an immunogen to produce polyclonal antibodies in rabbits and monoclonal antibodies in mice with good immunological properties through animal immunization and cell fusion assays.Double antibody sandwich ELISA detection method for BBI was established based on the two high-quality antibodies from different immune systems,and colloidal gold immunochromatographic strips for BBI detection was prepared based on monoclonal antibodies.（1）12-week-old New Zealand white rabbits were immunized by BBI.After the immunizations,the blood was collected from the heart and processed to separate and purify the serum,anti-BBI rabbit polyclonal antibodies were obtained and appraised the titer and sensitivity by ELISA methods.The results showed that the antibody titer was 1:1.024×10⁵,and the half-inhibition concentration(IC₅₀)was 306.76ng/mL.（2）BALB/c mice were immunized by BBI,and the mice that can produce antiserum with the highest titer and best sensitivity were selected for cell fusion.After repeatedly screening,two hybridoma cell lines that stalely secrete antibodies were obtained,named as 3B7-B10 and 7G7-D11.After prepared by inducing ascites in vivo,monoclonal antibody immunological characteristics were identified.The titers of the two monoclonal antibodies were higher than 1:4.096×10⁵,the half-inhibition concentrations(IC₅₀)were 83.07 ng/mL for 3B7-B10 and 186.81ng/mL for 7G7-D11,the subtypes of the two mAbs were both IgG1,and the affinity constant of 3B7-B10was 1.13×10⁸L/mol.The results of cross-reaction test and western blot assay showed that the two mAbs had very strong specificity.（3）Based on rabbit polyclonal antibody and murine monoclonal antibody（3B7-B10）,the double antibody sandwich ELISA detection method for BBI was successfully established.The method has strong reliability,the linear relationship between the concentration of BBI and the OD_450nm value in the standard curve is good.The linear regression equation was y=1.3836x-1.9496,R²=0.9795,the linear range is31.2¹000ng/mL,the detection limit is 31.6ng/mL,and the recovery rate is81.38%¹00.75%%with good specificity.（4）Based on the selected murine monoclonal antibody with excellent immunological characteristics,combined with colloidal gold immunochromatographic test paper technology,colloidal gold immunochromatographic strips for BBI detection was prepared,the visual and machine-readable detection limits were 0.5μg/mL and0.23μg/mL respectively.The accuracy and specificity of the test strip were identified,the results demonstrated that the strip has highly specificity,good repeatability and stability and can be used for rapid detection of BBI.