Preparation Of The Monoclonal Antibodies Againstβ-fructofurnosidase And Establishment And Preliminary Of The ELISA Method For Honey Adulteration Detection

Honey is nectar from the flowers of flowering plants collected by bees from the brewing of honey in the hive. Honey is also rich in various vitamins, minerals and amino acids, Addition to glucose, fructose. Its sweet taste, and monosaccharide which doesn’t digested can be absorbed by the body for women, young, especially the elderly good health, so it is a nutrient-rich natural nourishing food. Honey is in great demand. Driven by profit, domestic honey adulteration has a very serious problem in recent years, endangering the health of consumers, and a serious impediment to the healthy development of China apiculture.There are many ways to detect adulteration of honey, such as Isotope ratio analysis, high performance anion exchange chromatography-pulsed amperometric detection High performance liquid chromatography, near-infrared spectroscopy, polarimetry and high performance capillary electrophoresis. However, these methods have some kinds of shortcomings, for example equipment requirements, complex operation, single type of identification. Immunological methods, especially enzyme-linked immunosorbent assay (ELISA) give us a new approach for the detection of honey adulteration because of its high sensitivity, specificity, speed, and easy to operate,The beta-fructofuranosidase is used to the hydrolysis of sucrose in the Food Industry. In the high concentrations of sucrose solution, the β-fructofuranosidase catalytic transfers glycosylation role. In the low concentration of sucrose solution, the beta-fructofuranosidase catalyzes the hydrolysis of sucrose to generate of the fructose and glucose.If fructose and glucose are mixed in honey, it will be difficult to detect. And it will be helping to determine whether there is forgery of the plant conversion of carbohydrate in the detection of beta-fructofuranosidase residues in honey.In this study, we aims to establish a rapid, sensitive, simple and cost β-fructofurnosidase enzyme-linked immunoassay method and used in plant transformation of sugars of honey adulteration detection. We manufacture Anti-β-fructofuranosidase monoclonal antibody prepared by hybridoma technique, And to establish the side of the double-antibody sandwich ELISA for honey in the beta-fructofuranosidase residues. After comparing different sources of Beta-fructofuranosidase, we determine the protein concentration and purity with coomassie brilliant blue G-250, BCA and SDS-PAGE. The results show that protein concentration was5.6mg/mL in every10mg invertase dissolved in1mL ultrapure water. Then it was used as immunogen to immunize the mice. By the hybridoma technique, we get9hybridoma cells which were named as1D6F2,8F8C5,8F8A9,1D6D12,8F8G3,8F8A6,8F8F10,1D6B10,1D6B11.four kinds of abdominal dropsy were made by injecting1D6F2,8F8C5,8F8A9,1D6D12tumor cells to abdominal cavity of mice, and their titers were all above1:16000. The best concentration of ascites titer and coated antigen was gained, which were1:16000and1:8000. McAb-1D6F2and McAb-8F8A9might direct to the different antigenic determinant and on this basis, we establish the method of double-antibody sandwich ELISA. After result shows that the ELISA has a good specificity, high sensitivity, and no significant cross-reactivity. This method was initially applied to the detection of honey samples. Do8-fold dilution of the samples for the detection of honey adulteration, within5-80ng/mL the scope, the standard linear equation:y=-0.2359x+0.6421, R2=0.9878.