| As one of the main food allergens reported by FAO, seafood and its products have been recorded by more and more people. Since little quantity of allergen can cause serious allergic reaction, it is important to explore effective detection methods of allergens in order to ensure food safety. Using protein chips, this study try to construct a analysis system based on main seafood such as shrimp, fish and clam. The contents included separation and purification of seafood allergens, antibody production, 3D protein array slides, biological detection probes for allergen, and optimization of detection methods. The results are as follows.1. Optimized the separation and purification methods using Ammonium sulfate precipitation by adding crude seafood allergen extracts to a certain concentration, pH value of ammonium sulfate solution. It is helpful to improve the accuracy of Ammonium sulfate precipitation. Then by cutting single band from Gel Electrophoresis, we have got seafood allergen with more than 95% purity.2. The study has optimized the preparation conditions for three-dimensional protein chip. Using atomic force microscopy (AFM) and X-ray photoelectron spectroscopy, we characterize the physical and chemical properties of modified chip surface. The results showed that using 3% amino-silane to modify glass, 0.1mol/mL NaIO4 for activating agarose in chip surface, we can form a uniform grid of three-dimensional structure, and surface activity derived aldehyde can help to improve the hydrophilic properties of the base and the formation of three-dimensional gel on the surface of chip.. These fixed elements can help to enhance the effect of immubolization.3. The study has got high titer polyclonal antibodies by immuning New Zealand rabbits with purified shrimp, fish, and clam allergen respectively. The titer of anti-serum was 1.5×10~6, 2.0×10~6 and 1.0×10~6. For monoclonal antibody, we have got totally nine monoclonal cells. After characterization, we found that 2 pieces of shrimp monoclonal cells were IgG1; 3 pieces of fish monoclonal cells were IgG1; 1 piece of clam monoclonal cells was IgG1; 1 piece of clam monoclonal cells was IgM; 1 pieces of clam monoclonal cells were IgA. Then the monoclonal antibodies were biotin-labeled to prepare seafood allergen sensitive detection probes.4. The best response conditions for seafood allergen detection were determined through response surface methodology. The results showed that detection limit of shrimp allergen was 20ng/mL with linear detection range of 20.0ng/mL ~ 20.0μg/mL; the detection limit of clams allergen was 40ng/mL with linear detection range of 40.0ng/mL ~ 40.0μg/mL; the detection limit of fish allergen was 20ng/mL with linear detection range of 20.0ng/mL ~ 20.0μg/mL.5. Seafood allergen detection chips performance were determined. The results showed that the intra-and interassay coefficient of variation (CV%) were among 5% -10%. No cross-reactions were happened in different allergens. Using steaming bread as food matrix, the recovery rate of these kinds of seafood were more than 75%; while in cow milk,the recovery rate of fish and shrimp allergens were more than 90%, for clams, it could reach to 80%.At present, the methods of detecting seafood allergens mainly concentrated in the traditional methods such as enzyme-linked immunosorbent assay. Seldom studies about the use of immune-chip on the detection of seafood allergens were indicated. In this study, combined with the characteristics of the allergens detection, the glass silde of three-dimensional chip was modified for the first time, which has improved the fixation effects of three-dimensional chip. On this basis, the detection method of seafood allergen was established and improved, which will provide a necessary technical support to the control of seafood allergens.
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