Quantitative Detection Method Of Tetrodotoxin And Okadaic Acid In Aquatic Products By Fluorescence Immunochromatography

Tetrodotoxin and okadaic acid are the most harmful marine toxins in aquatic products,which are continuously accumulated in aquatic products,thus threatening the safety of human life.ELISA kits are often used for the detection of marine toxins,which is difficult to achieve rapid on-site detection of multiple toxins.Therefore,the construction of multi-toxin on-site rapid detection technology has important application value for the safety and supervision of aquatic products in coastal areas.In order to quantitatively detect OA and TTX toxins in aquatic products,time-resolved fluorescent microspheres were used as signal reporters in combination with chromatography technology to complete the construction of TTX immunochromatography method and TTX-OA simultaneous detection method.On this basis,after the recombinant vector was constructed and integrated into E.coli,the OA single-chain antibody was induced to express in the cells,and simple screening and identification were carried out.The main research contents and results are as follows:（1）The TTX fluorescence immunochromatography method was established to realize the quantitative detection of TTX in aquatic products.Biotin and artificial antigens prepared by the formaldehyde method were sprayed on different positions of the test strip membrane as the control line and the detection line,respectively.The antibody-microsphere binding probe was prepared and characterized,and the TTX fluorescent test strip detection technology was constructed.Within the linear range of 0.5 ng/m L to 40 ng/m L,the linear fitting equation of TTX was y=-0.2668x+0.57365（R²=0.9940）,and the limit of detection（LOD）was 0.047 ng/m L.The recoveries of the spiked aquatic products samples were between 97.08%and 101.86%,and the relative standard deviations were all lower than4.70%.The method has the advantages of low cost and high sensitivity,which is beneficial to the rapid quantitative detection of TTX.（2）Chemical-based preparation of OA antigens and expression of single-chain antibodies.The OA artificial antigen was synthesized by activated ester method and verified by infrared spectroscopy.In the infrared spectrum,the protein showed characteristic peaks of carboxyl groups at 3422 cm^-1and 1655 cm^-1.The wave number of C=O of-CONH-in the antigen is 1661 cm-1,and the wave number of-NH is 3431 cm^-1.The above indicated that the OA complete antigen was successfully synthesized.OA single-chain antibody is a new type of specific immune element synthesized based on genetic engineering technology,which is expected to replace the preparation of antibodies in animal experiments that may cause ethical problems.The expression vector of the target gene was constructed and integrated into Escherichia coli to induce the expression of the target gene.In the ELISA identification result of the product,the ratio of positive to negative control was higher than2.1（P/N>2.1）,and the test result was positive,which proves the specificity of the antibody.（3）A fluorescence immunochromatography technique for simultaneous detection of TTX and OA was established to realize the detection of TTX-OA in aquatic products.In this study,the coating antigens of TTX and OA,and biotin-bovine serum albumin were used as detection and control lines,respectively.Antibody-microsphere probes were synthesized and characterized.An immunochromatographic test strip for simultaneous detection of TTX and OA in aquatic products was established.Under the conditions of p H 7,antibody microsphere ratio of 1:10,and reaction time of 12 min,the linear fitting equation of TTX was Y_TTX=-0.2624X+0.8247（R²=0.9916）,LOD=0.167 ng/m L.The linear fitting equation of OA was Y_OA=-0.4333X+1.1133（R²=0.9849）,LOD=1.624 ng/m L,the coefficient of variation of this method is less than 10%,and the recovery rate is between 84.48%and111.90%.The test results of this method are consistent with the test results of the instrument method,and the test strip can be stored for more than one year when the storage condition is 4℃。...