| Fish is an important source of food for humans,not only delicious but also nutritious.Fresh fish is rich in 15%to 20%of full-price protein,1%to 10%fat,and is rich in important unsaturated fatty acids.China is a large aquaculture country,accounting for more than 70%of the world's aquaculture production.Fish and its products are rich in nutrients and are among the eight foods recognized by FAO and WHO to cause allergies in humans.Among them,parvalbumin?PV?in fish meat is regarded as the main allergen,so it is of great theoretical and practical significance to carry out detection of strong specificity and sensitivity for small albumin in fish and its products.The research in this paper mainly includes the preparation and identification of monoclonal antibodies against parvalbumin,the preparation of gold-magnetic composite nanoparticles and the establishment of a small-protein gold-magnetic immunochromatographic assay.The specific research content is as follows:1.Preparation of small albumin monoclonal antibodyFour BALB/c mice were immunized with high-purity PV,and the mouse antiserum titer was measured by indirect ELISA to obtain the mouse with the highest titer.The spleen cells of the immunized mice were subjected to cell fusion with SP2/0myeloma cells to obtain hybridoma cells.The positive wells were screened by indirect ELISA and subcloned three times by limiting dilution to obtain homozygous hybridoma cell lines.Monoclonal antibodies were prepared in large amounts by inducing ascites in mice.Monoclonal antibody ascites was purified by caprylic acid-ammonium sulfate precipitation method.The purity of monoclonal antibody was characterized by SDS-PAGE gel electrophoresis.The specificity and antibody concentration of monoclonal antibody were detected by Western-blotting and Coomassie blue staining.The antibody has good specificity,and the concentration of the monoclonal antibody is 0.73 mg/mL.Finally,the PV monoclonal antibody subclass is detected by the commercial kit as the IgG1 type.2.Qualitative detection of parvalbumin gold magnetic immunochromatographic test stripsThe aluminized magnetic particles Fe3O4-PEI were prepared by one-pot method,and then two layers of Au particles were coated on the surface to prepare gold magnetic?Fe3O4/Au?composite particles.The gold-magnetic composite particles were round in shape and uniform in particle size.The particle size is about 80 nm.The gold magnetic composite particles were coupled with the monoclonal antibody of small albumin to prepare a gold magnetic immunoprobe,and the optimum pH value,antibody dosage,reaction time and buffer system of the coupling of the gold magnetic probewereoptimized.Theparvalbumingoldmagneticprobe immunochromatographic test strips were assembled according to the test strip assembly method,and the sample pad,the bonding pad,the material selection of the nitrocellulose membrane,the optimal formulation of the treatment liquid and the optimal treatment conditions,T were explored./C line optimal line concentration and other test strip assembly conditions are optimized.The standard product was tested.The results showed that the prepared gold magnetic immunochromatographic test strip had a good qualitative function.The minimum detection concentration of the visual detection was 4 ng/ml,and the detection specificity and detection stability were good.3.Quantitativedetectionofparvalbumingoldmagnetic immunochromatographic test stripsBy establishing a quantitative method of T/C ratio,the detection sensitivity of gold magnetic gold immunochromatographic test strips was improved.At the same time,combined with the actual situation of the grassroots work,seven kinds of seven kinds were compared from the aspects of ease of operation,time consumption of pretreatment and recovery rate.Advantages and disadvantages of pretreatment methods for fish samples.On this basis,a rapid detection method for quantitative analysis of small albumin in fish meat by gold magnetic immunochromatography test strip T/C ratio method was established. |
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