| Food allergy is one kind of allergic diseases in humans, is an adverse health effect arising from a specific immune response that occurs reproducibly on exposure to a given food or food components. Food allergy can triggering tissue injury of the body’s digestive system, respiratory system, circulatory system and integumentary system, it can even cause fatal anaphylactic shock. Thus, food allergy has been regarded as one of the food safety issues that affect public health nutrition. As people’s increasingly attention to the problems of food allergy, some rapid and accurate detection methods for food allergens have been developed. The immunological methods have become a research hotspot, because they have characteristics of high sensitivity and strong specificity, and can carry on the qualitative and quantitative detection. As one kind of important food crop, soybean is used widely, and plays an important role in human and animal nutrition; but soybean is one of the eight allergic source food, contains a variety of allergenic proteins, can cause infants and young animals to appear allergic reaction. In this research, the research object are glycinin and β-conglycinin as two main soybean allergenic proteins. Based on the rabbit polyclonal antibody(p Ab) and mouse monoclonal antibodies(m Ab), which have good immunological features, the double antibodies sandwich ELISA kit and colloidal gold immunochromatography test strip have been developed for rapid detection of glycinin and β-conglycinin, respectively. On the one hand, they can be applied to rapidly supervision the soybean allergenic component labeling information, to further improve soy allergy consumers’ food safety; On the other hand, they can be used as an effective tool for the study of soy allergenic protein desensitization method to identify the feasibility and effectiveness of desensitization method. This study’s main research results are as follows:(1) Using isoelectric point precipitation to separate glycinin and β-conglycinin from defatted soybean meal. New Zealand white rabbits were immunized by glycinin and β-conglycinin, respectively, with the neck multi-point subcutaneous injection. After four injections, collected blood and separated serum. Using saturated ammonium sulfate precipitation to purify glycinin rabbit p Ab and β-conglycinin rabbit p Ab. Appraised by ELISA methods, the antibody titers of glycinin rabbit p Ab and β-conglycinin rabbit p Ab were 1:1.024×106 and 1:5.12×105, and they both have good sensitivity and specificity.(2) BALB/c mice, which were 6~8 weeks old, immunized by glycinin and β-conglycinin, respectively. Two mice were chosen for cell fusion because their antiserum have high titer and good sensitivity. Applying cell fusion technology to establish hybridoma cell lines. Through the screening of positive hybridoma, we obtained one hybridoma strain that stablely secrete glycinin m Ab, named as 2C3; three hybridoma strains that stablely secrete β-conglycinin m Ab, named as 1G6, 7E7, 8C11. Then these four m Ab were prepared by mice induce ascites. The subtype of these four m Ab are all Ig G1 type. Via indirect ELISA, the titers of them were 1:5.12×105, 1:2.56×105, 1:1.024×106 and 1:5.12×105, respectively; via indirect competitive ELISA, the half inhibitory concentration of them were 498.7 ng/m L, 844.2 ng/m L, 479.4 ng/m L and 889.6 ng/m L, respectively; via saturation ELISA method detected the affinity constant of them were 4.27×109 L/mol, 3.29×108 L/mol, 1.34×109 L/mol and 3.48×108 L/mol, respectively. Cross reaction test and Western blot test showed that all four m Abs have strong specificity.(3) Based on the preparation of rabbit p Ab and mouse m Ab, using double antibody sandwich model to establish glycinin and β-conglycinin ELISA detection method. Glycinin double antibody sandwich ELISA detection kit was developed by using 2C3 m Ab and glycinin rabbit p Ab, the limit of detection was 16.3 ng/m L, the linear detection range was 15.625~2000 ng/m L, the recoveries of glycinin spiked in milk powder samples were 80.5%~88.9%, the variation coefficient was less than 15%. β-conglycinin double antibody sandwich ELISA detection kit was developed by using 7E7 m Ab and β-conglycinin rabbit p Ab, the limit of detection was 15.4 ng/m L, the linear detection range was 15.625~2000 ng/m L, the recoveries of β-conglycinin spiked in milk powder samples were 83.4%~90.9%, the variation coefficient was less than 15%. These two ELISA kits have no cross reaction with other common food allergenic proteins, have good specificity and stability. The validity of these two ELISA kits in 4 ℃ was above three months.(4) Based on the colloidal gold immunochromatography assay, the colloidal gold immunochromatography test strips for glycinin and β-conglycinin rapid detection were firstly developed by using the rabbit p Ab and mouse m Ab, respectively. Visual and machine-readable LOD of glycinin test strip were 100 ng/m L and 46.1 ng/m L, respectively; visual and machine-readable LOD of β-conglycinin test strip were 200 ng/m L and 152.8 ng/m L, respectively. The recoveries and variation coefficient of these two test strips were both within the normal range. These two test strips have no cross reaction with other common food allergenic proteins, have highly specificity, good repeatability and stability. The validity of these two test strips in normal temperature by sealing dry preservation was above three months. |
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