| Docosahexaenoic acid(DHA)is a major component of the growth and maintenance of nervous system cells.DHA cannot be synthesized in the human body and can only be taken from the diet.With the universal application of microalgal source DHA in food addition,the quality and safety of microalgae source DHA has gradually attracted the attention of consumers.However,it is understood that,although research reports on DHA and commercial products have emerged endlessly,gold standard rapid test products for detecting DHA content of microalgae have not yet been produced on the market.Therefore,this topic is based on previous studies of hapten coupling techniques,antibody preparation techniques,etc.,to study the preparation of microalgal-derived DHA coupling antigens and antibodies.The application of DHA antigen antibody in colloidal gold immunochromatography was discussed.In this study,the immune antigen DHA-BSA and the coating antigen DHA-OVA were synthesized by carbodiimide and mixed anhydride methods.The hapten DHA was successfully coupled to the carrier proteins BSA and OVA by protein electrophoresis,infrared spectroscopy,and ultraviolet absorption spectroscopic analysis.The coupling ratios of the immunogenic antigens were4:1and17:1,and the appropriate coupling ratio range was achieved.Balb/c mice were immunized four times with the immunizing antigen,and finalBalb/c mice were immunized four times with the immunizing antigen and sera were tested for potency.Finally,20μg/ml of coated antigen was used for coating,and 5%skim milk powder was used for blocking.The titer of antiserum was 128,000.The IC50 of the antibody was determined to be 40.44μg/ml by blocking ELISA.The antibody was purified with the protein purification column protein G.The concentration was 34.27 mg/ml before purification and 0.917 mg/ml after purification.After SDS-PAGE protein identification,the antibody purification effect is better.After treatment with reductive treatment solution,there are only two bands of heavy chain and light chain.In this study,trisodium citrate reduction method was used to prepare colloidal gold solution.By adjusting the amount of trisodium citrate,the best state of colloidal gold firing was determined.When the ratio of trisodium citrate to chloroauric acid was 1.5:1,the prepared colloidal gold solution had clear color and good stability,and its maximum absorption wavelength was 521 nm and the particle size was measured after scanning at full wavelength of visible light,about 20 nm in size,suitable for the preparation of gold standard antibody.During colloidal gold labeling of antibodies,the acid-base environment prepared by the gold-labeled antibody was indirectly regulated by adjusting the amount of K2CO3 added,and the amount of antibody was adjusted so that the gold-labeled antibody was stable and active.By visual observation and visible light spectrophotometry,it was determined that the optimal amount of K2CO3 was 4μl,and the optimal amount of antibody was 13μg.The treated gold-labeled antibody was coated on the gold standard pad at 6 μl/cm,and the 50 mg/ml coating antigen and 0.2mg/ml goat anti-mouse IgG were coated on the NC membrane at positions T and C respectively at 1μl/cm.Complete colloidal gold immunochromatographic test strips,negative test with 0.02M PBS solution,positive test with diluted DHA standard,and preliminary investigation of colloidal gold test card for DHA from microalgae. |
PDF Full Text Request