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The Preparation of Anti-ovalbumin Monoclone Antibody and the Quantitative Determination of Ovalbumin

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Tutor: MaHongMing
School: Ocean University of China
Course: Aquatic Products Processing and Storage Engineering
Keywords: Monoclone antibody,Ovalbumin,Gliadin,ELISA
CLC: R155.5
Type: Master's thesis
Year:  2010
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Abstract:
Allergic disease, affecting nearly 1/4 of the population in the world, of which 3% to 5% is food allergy, is one of the three diseases classified as the focus of prevention and cure in the 21st century by the World Health Organization. On July 19,2005, the U.S. Congress passed an act, Food Allergen Labeling and Consumer Protection Act (FALCPA), to require food manufacturers clearly indicate whether the food contains eight most common food allergens or not. Eight common food allergens (egg, milk, soy, peanuts, wheat, tree fruit, fish and shellfish) are detected by the import and export inspection and quarantine system, but lacking of appropriate standards and methods. This article dedicated to the research of detection methods for eggs and wheat allergen and to the theory basis for the development of appropriate standards.Ovalbumin is the major protein and one of the major allergens in chicken egg. A double antibody sandwich enzyme-linked immunosorbent assay for quantitative analysis of common allergens ovalbumin in foods involoving the preparation of hybridoma cell lines which could secrete monoclone anti-ovalbumin antibody was established in this article. In the study, BALB/c mice were immunized three times with purified ovalbumin adding adjuvant or not until the serum titer achieved 10-5, and the mice spleen cells and myeloma cells SP2/0 were fused as the routine cell-fushion technology after three days of booster. Positive cells were screened with indirect ELISA by coating purified ovalbumin, positive hybridoma cells were cloned through three times of limiting dilution. Finally, six strains of hybridoma cells secreting special monoclone antibodies had been obtained, named OVA-1C6, OVA-2F9, OVA-2D8, OVA-2D2, OVA-3H6, OVA-3G9 and OVA-2D8. The titers of their cell culture supernatants were not degraded in spite of cell resuscitation, which were 1:50000,1:30000,1:30000,1:10000,1:10000 and 1:5000, respectively. They were all IgG, in which the hypotype of OVA-1C6 was IgG2b, OVA-2D8 and OVA-2D2 were IgG2a, OVA-2F9, OVA-3H6 and OVA-3G9 were IgGl. These monoclone antibodies could react with ovalbumin, and cross reactivity were not observed with bovine serum albumin, porcine serum and rabbit serum. Selecting two monoclone antibodies of highest antibody titers were OVA-2F9 and OVA-3G9, with which were prepared ascites. After the purification of ascites, monoclone antibodies were separately labelled with horseradish peroxidase, and OVA-2F9 was labelled successfully, named HRP-2F9. The method of double antibody sandwich ELISA for ovalbumin was successfully established by optimizing parameters. The optimum concentrations of coating antibody OVA-3G9 and enzyme labelled antibody HRP-2F9 by matrix titration assay were 1:200 and 1:1000 respectively. The optimal linear range was 97.66ng/ml to 6250ng/ml and the coefficient of correlation was¡Ý0.995. The minimum detection limit was 97.66ng/ml, approximately 0.1 ppm, corresponding to 1.95ppm in original foods. The recovery rate for the accuracy test was 91.92%-100.34%. The coefficients of variation for precision assay of intra-assay and inter-assay were 1.22%-5.14% and 3.17%-6.83%, respectively. The set of kit could be placed freezer in 4¡æfor one month. No cross reactions were observed with chicken, pork and beef, but it was specific to ovalbumin of egg such as henegg, duck¡¯s egg, and common quail egg. The feasibility of the kit was tested by the recovery of ovalbumin in foods and by comparing test with the abroad RIDASCREEN ei/egg white proteins test kit. Results tested by above developed test kit are basically matched with abroad kit for ovalbumin, and detection sensibility of self-made test kit is ten times higher than lppm of abroad kit.Gliadin is the major allergens of wheat and other cereals, accounting for about 40% of the total storage proteins of wheat seeds, and it can be one of detect targets for wheat. A method for quantitative analysis of gliadin from wheat in foods by sandwich ELISA was established in this paper. In this study, BALB/c mice were immunized at least three times with gliadin from wheat dissolved in 65% ethanol, and from that immune serum was as the positive serum control. By matrix titration assay, the mouse anti-gliadin monoclone antibody was used as a coating antibody in the concentration of 2¦Ìg/ml, and a horseradish peroxidase-conjugated anti-gliadin as an enzyme-labeled antibody in the optimal working dilution of 1:8000. The double antibody sandwich ELISA method for gliadin and the development of its test kit were established by selecting optimal blocking reagent. Under the optimal conditions of antibodies concentrations, the appropriate blocking solution was selected, which is PBST with 10% fetal bovine serum, then the standard curve was fitted by a series of concentrations of gliadin solution as standards. The feasibility of the kit was tested by the recovery of gliadin in foods. The optimal linear range was 19.53ng/ml to 5000ng/ml and the coefficient of determination R2 was> 0.99. The quantitative limit of detection was 19.53ng/ml, which is corresponding to 7.81ppm in food samples. The recovery rate for the accuracy test was 93.57%-105.72%. The coefficient of variation for precision assay were 3.07%-5.91% in the same test and 0.26%-5.24% among three tests. Cross reaction was only observed with barley. The set of kit could be placed freezer in 4¡æfor two weeks. It is suitable for quantitative determination of gliadin in foods.The double antibody sandwich ELISA methods for ovalbumin and gliadin were established respectively. Both of methods are sensitive, accurate, specific and stable. Sensitivity of ovalbumin ELISA test kit for quantitative detection is ten times higher than the import kits, detecting costs is only about half of the imported products, and the technical parameters of kit is better than or equivalent to the level of similar foreign products, meeting the daily requirements of import and export inspection and establishing a theoretical foundation for developing appropriate methods and standards of domestic.
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