| Cefquinme is the first fourth-generation cephalosporin antibiotic specially used for animals.Compared with the third-generation cephalosporin antibiotics,it has some characteristics with broader antibacterial spectrum,higher bioavailability,stronger antibacterial activity and stronger penetration of cell membrane.At present,it is generally used to treat mastitis and endometritis in cow and respiratory diseases in cow and in pig.Because cefquinme is used in a unreasonable way,it will result in cefquinme residues in food of animal origin,and bring potential harm to human health,even more,cause food safety and public health issues.The European commission for drug evaluation has made a clear regulation on the maximum residual amount of cefquinme in edible tissues of cattle and pigs.Nowadays,the main detection methods that it has multi-pretreatment and the higher cost and interference factors and unstable sensitivity,was widely used in the practical work hard,however,the immunological assay has the advantages of quick and easy and low-cost that is widely used in the prophase work of screening,at the same time,improving the efficiency and enhancing the reliability of the later experimental results.At present,there are few reports about the immunological detection of cefquinomeat home and abroad.In this experiment,according to the structural characteristics of cefquinoxime,artificial antigen was synthesized;By the cell fusion technique.to produce Anti-cefquinome monoclonal antibody with high specificity and sensitivity;to establish a stable-secreted cefaqume monoclonal antibody cell line in order to provide a good foundation in subsequent immunological detection.1.Synthesis and identification of cefquinoxime complete antigenBy means of carbon diimide method,the immunogen CEF-BSA and the coated antigen CEF-OVA were obtained by coupling cefquinme with carrier proteins(BSA and OVA).After the UV scanning,SDS-PAGE analysis and immunological identification,the results indicate that the immunogen and the coating antigen were successful preliminary.The protein concentrations of CEF-BSA and CEF-OVA were measured by BCA kit at 4.2 mg/mL and 4.6 mg/m.2.Preparation of cefquetrime monoclonal antibodyBALB/c mice were immuned by artifical antigen CEF-BSA and collected blood and centrifugation,its serum titer of immunity was 1:16000.Two positive hybrid tumor cell lines were screened by cell fusion technology and limited dilution method,which were named as 2E5 and 4G7 respectively.The cells were injected into the abdominal cavity of mice toget the ascites monoclonal antibody.Purified and identified y Protein GHP affinity chromatography column,the titer of 2E5 monoclonal antibody was 1:32000.The results of cross experiment showed that the monoclonal antibody had no cross reaction with cefalexin.ceftiofur,cefphalothin,lomefloxacin and penicillin,and it had good specificity3.The establishment of ELISA detection methodThe purified ascites was established an indirenct com petitive ELISA method for the detection of CEF residues.The optimal working concentration of the antigen and antibody for the monoclonal antibody was found out,the optimum concentration of the coating antigen was 1:6000 and the optimum concentration of the antibody was 1:8000.When the CEF concentration was between 50 to 5000 ng/mL,the linear regression of 2E5 monoclonal antibody was y=0.2854 x-0.189,IC50=258.42 ng/mL,LOD=5.87 ng/mL,LOQ=20.54 ng/mL.4.The addition and reclamation test of CE F in the Fresh milkIn the recovery experiment of CEF in the fresh milk sample,the interference of matrix in the milk sample can be basically disappeared when the milk sample is diluted 8 times.The concentration range of CEF from 20 to 2000 ng/mL,the linear regression was y=0.4493x-0.5184(R2=0.9895),IC50 was 183.76 ng/mL,LOD=15.30 ng/mL,and LOQ=106.03 ng/mL,High(50),medium(500)and low(2000)of CEF standard solution to do the recovery experiment,and the recovery rate of milk sample was detection between 82.73%?102.15%. |
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