Preparation Of Monoclonal Antibody For Nitrofurazone Metabolite SEM And Study On Quantitative Test Specimen Of Fluorescence

Furacilin metabolite SEM are carcinogenic,carcinogenic and have other side effects on humans,On March 22,2010,the Chinese Ministry of Health included the addition of nitrofurazone to blacklist of non-food substances that could be illegally added.At present,the detection technology of toxic and harmful substances in animal food mainly includes chromatograpHic technology and immunoassay technology.Sample processing of chromatograpHic technology is cumbersome and time consuming,requiring expensive equipment and operations.Immunoassay techniques mainly includes enzyme-linked immunoassay(ELISA)and colloidal gold(colloidal gold test strip).ELISA method has the characteristics of high sensitivity and high throughput,but the detection time is longer.Colloidal gold method is characterized by fast(10-15 minutes)and easy to operate,but the detection sensitivity is lower,5-10 times worse than the ELISA method.In order to establish a fast,highly sensitive,high-throughput immunoassay method,In this study,SEM antigen and its monoclonal antibody were prepared on the basis of SEM immunological properties,and a SEM fluorescent quantitative detection strip was developed.The main research results are as follows:(1)Synthesis and identification of furacilin metabolite derivant of artificial antigenIn this study,SEM and NOCP(4-(3-aldehyde-4-nitro-pHenoxy)-butyric acid)were used by organic synthesis to prepare hapten(NOCPSEM)with similar structure to 2-NPSEM,and the hapten was identified by mass spectrometry and nuclear magnetic resonance.The immunogen NOCPSEM-BSA and the coated NOCPSEM-OVA were prepared by coupling the synthesized hapten with BSA and OVA respectively by the activated ester method,And identified by UV scanning.(2)Preparation and Identification of Monoclonal Antibody Against 2-NPSEMThe monoclonal antibodies with high purity and specificity were prepared by immunizing BALB / C mice with synthetic artificial antigen NOCPSEM-BSA.The concentration of NOCPSEM-OVA and the concentration of antibody were optimized,The titer of anti-2-NPSEM monoclonal antibody was finally determined to be 2560000,The IC50 value was 0.23 ppb,The cross-reactivity with structural analogues(2-NPAMOZ,2-NPAOZ and 2-NPAHD)was less than 0.1%(3)Development of fluorescent quantitative detection test stripThe fluorescence microspHere activation system,dilution ratio of fluorescent microspHere,the concentration of test line and quality control line,the type of treatment solution and the dilution of sample were optimized respectively,A method for detecting 2-NPSEM fluorescence quantitative test strip was established.The sensitivity of the test strip was 0.2 ppb;the detection time was 15 min;When the sample was added with 0.5 ppb,the recovery was 80-136%;The false positive rate of false leave was 0;the cross reaction rate with structural analog was less than 0.1%;Test strip of the batch and the inter-assay coefficient of variation are less than 5%;While heated at 50 ℃ for 60 days,Test strips had no change in performance.As the results showed,A method for quantitative determination of SEM residues by fluorescence spectrometry was developed,Compared with the ELISA method,the detection time was shortened from 75 min to 15 min,and the quantitative detection,and the quantitative detection could also be realized.Compared with the colloidal gold method,while the detection method maintained the same rapid premise,its sensitivity increased by about 5 times.The fluorescence quantitative test strip can meet the needs of the actual sample testing and also lay the foundation for the assembly of the commercial SEM quantitative test strip.