Preparation Of Monoclonal Antibody Against Major Allergen Of Fish And Construction Of Its Enzyme-linked Immunosorbent System

Fish and its products are rich in nutrients,and its dairy products are considered to cause human one of the eight kind of food allergies by FAO and WHO.Parvalbumin（PV）of fish and its products are regarded as the mainallergens.Therefore,there is a great theoretical and realistic significance for the strong specificity,high sensitivity of detection of parvalbumin in food products.In this paper,we mainly studied including preparation and appraisal of monoclonal antibody against PV,preparation of gold magnetic composite nanoparticles and the enzyme-labeled antigen,and development of gold magneticenzyme-linked immunoassay for detecting allergens PV in fish and its products.The concrete research content is as follows:1.Preparation of fish major allergen parvalbuminThe allergen albumin was purified by ammonium sulfate fractionation method from fresh carp flesh,and the purity of the allergen was detected by SDS-PAGE electrophoresis.Finally,the samples of parvalbumin were freeze-dried and stored in the-20 ℃refrigerator.2.Preparation of monoclonal antibody against PVFour BALB/c mice were immuned with the high pure PV respectively,and indirect ELISA method was used to test the titer of antiserum of mice to obtainthe highest antiserum titer of mice（the titer of PV was 1:256000）.Cell fusion was carried out by immunized mice spleen cells and SP2/0 myeloma cells,so as to obtain hybridoma cells.The indirect ELISA method was used to screen the positive hole,and through 3 subcloning,Secreting homogeneous hybridoma cell lines were obtained by limited dilution method.Finally,a large number of monoclonal antibodies were prepared by the method of induced ascites in mice.3.Appraisal of monoclonal antibody against PVMonoclonal antibody was purified using caprylic acid-ammonium sulfate precipitation joint.The purity of the obtained monoclonal antibody was characterized by SDS-PAGE electrophoretic,and Coomassie brilliant blue staining was used to detect the concentration of PV antibody.The results showed that PV antibody has good specificity and the concentration of purified PVm Ab was 300 ug/m L.Finally,The commercialization kit showed that the PV monoclonal antibody was type Ig G1.4.Synthesis of gold magnetic composite and the enzyme-labeled antigenAmino-functionalized Fe₃O₄-PEI composites,which were synthesized by the onepot solvothermal method.After two layers of Au particles were coated on the surface,of preparing gold magnetic（Fe₃O₄/Au）nanoparticles.Shapes of gold magnetic composite were round,uniform,whose particle size were about 150 nm.PV were labeled with horseradish peroxidase（HRP）by the sodium periodate method,which was used for the establishment of the subsequent magnetic enzyme immunization system.5.Development of gold magnetic enzyme-linked immunoassay for detecting allergens PV infish and its products.After the gold immunomagnetic probe and PV enzyme labeled antigen and HRPCAP were modified as the optimum conditions described above,they were mixed with PV standard solution to establish gold magnetic enzyme linked immunosorbent assay system.And the feasibility of the system in the detection of allergens PV infish and its products was investigated preliminarily.The results showed that there was an excellent linear tendency between logarithm of PV concentration and the inhibition rate in the measured range of 5.5 ng/m L-289 ng/m L,the standard curve was y=34.96x-6.0357,R² =0.9979,IC₅₀ was 40.1 ng/m L and LOD was 2.8 ng/ m L.In addition,the detection system was evaluated,including precision,specificity and stability.The results showed that there was a good precision with intra-assay coefficient of variation of 4.3 %-9.6 %,inter-assay coefficient of variation of 5.5 %-11.7% in detecting PV.The crossreactivity rates of the system with other major proteins in food was less than 1 %,which had a good specificity.Finally the stability of gold magnetic probe was in about 15 days.This method can improve the sensitivity and specificity,and hasan important practical application value in food safety detection.