| ?-Conglycinin is an important storage protein in soy.Soybean is one of the eight main allergens,which can cause people food allergies.There is no effective methods of prevention and eradication for food allergy in clinic,for which the only safe and effective way is to avoid eating and contacting allergens.For the potential allergic patients of soybeans and soybean and its by-product processing enterprises,it is particularly important to establish a quantitative detection method for?-conglycinin.Due to the high sensitivity,high affinity,and fingerprint resolution,the surface-enhanced Raman scattering technique can enhance the signal strength by 6 to 10 orders of magnitude.Therefore,based on the preparation of?-conglycinin antibody,the double antibody sandwich method for rapid detection of?-conglycinin was assembled,and a new Raman enhanced immunoassay method using colloidal gold as active substrate was established.The detection of allergens in allergic consumers'foods provides a convenient and quick detection tool.The main research content is as follows:1.Natural?-conglycin was immuned to BALB/c mice for many times.Fives times later,polyclonal antibody titer by indirect ELISA method was as high as 1:12800,which IC500 value was the concentration of 718 ng/mL.With wheat germ globulin and Sesame protein,the cross reaction rates were less than 0.2%.The cross reaction rate with peanut protein was 5.6%.And the cross reaction rate with glycinin was 8%.After several immunization,it was found that the effect of 60?g immune dose was better than 50?g on mouse polyclonal antibody preparation.2.The high-valent and strong-sensitive mouse spleen cells and myeloma cells were fused into hybridoma cells by cell fusion technology,and the cell lines producing specific antibodies 3C9 and 3D11 were obtained by screening and subcloning,and induced by in vivo induction method.A large number of monoclonal antibodies were prepared to obtain two monoclonal antibodies,3D11 mAb and 3C9 mAb.The two monoclonal antibody subtypes were identified as heavy chain IgG1 type,light chain Kappa type,and had good specificity,and all reacted strongly with?'subunit;indirect ELISA determined that the titer of 3D11 mAb reached 1:1.28×105.The IC50 value of the half inhibitory concentration was 911 ng/mL,and the affinity constant was 9.6×109 L/moL.The titer of 3C9 mAb was 1:2.56×105 by indirect ELISA.The IC500 value was 2.91?g/mL,and the affinity constant was 1.42×1011 L/moL.The antibody was purified by n-octanoic acid and ammonium sulfate precipitation method,and the purity of the monoclonal antibody was increased to 72%after purification.3.A rabbit polyclonal antibody against?-conglycinin was used as a detection antibody and a double antibody sandwich immunochromatographic test paper method using a 3D11mAb as an immobilized capture antibody.A Raman immunoprobe was prepared by modifying colloidal gold with 4-aminothiophenol and binding to the antibody to establish a rapid detection method for surface-enhanced Raman lateral immunochromatographic test paper.The SERS-LFA method can detect the?-conglycinin content in the sample from 160 ng/mL to100?g/mL,which satisfies y=65.1360x+113.9210,R2=0.9636,and the detection limit is 32ng/mL.This assay could provide a valuable tool for sensitive determination of?-conglycinin from different samples. |
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