| Doxycycline(DC) is a kind of alkaline broad-spectrum and semisynthetic tetracycline antibiotics, it was used in prevention and treatment of poultry infectious diseases, as well as the food additives. However, in practice, illegal administration leads to doxycycline residue in animal tissue and by-products, it damages to food safety and human health.We can adopt traditional detective method of instruments, it consists of HPLC with UV detection method, liquid chromatography tandem mass spectrometry method, thin layer chromatography analysis method. It needs expensive equipment, complex sample pre-treatment process, and professional staff. These minus points lead to this detective method was unsuited for large number of sample testing. Microbiological assay method is time consuming, and the specificity is lower, it affects the accurate examination result. The detection method of ELISA is dependent on on the basis of antigen antibody reaction, its specificity is strong, sensitivity is higher, and it is sample, efficient and flexible. So it is fit for a vast number detection.Using the method of Azo benzoic acid and EDAC composes Doxycycline complete antigen, it can immune BALB/c mices after dialysis and ultraviolet scanning. By techniques of monoclonal antibodies, ELISA and producting ascites to prepare monoclonal antibodies against doxycycline with high titer and specific. And we establish the indirect competitive ELISA method to detect doxycycline residue.1. Synthesis and characterization of DC complete antigensDC belongs to semi synthetic tetracycline drug and is the derivatives of Hydrogenation and four benzene. Its molecule contains hydroxyl groups. By the method of Azo benzoic acid to transformate DC molecule, and then using the method of EDAC to conjugate with carrier protein(BSA, OVA), at last it forms solid molecular spacer arm and amide bond which is complete antigen synthesis. The complete antigen was demonstrated by UV spectrum scanning, and then after immunization, cell fusion and ELISA detection method, the result show that the two complete antigen was successful. The protein concentrations of DC-BSA and DC-OVA were 2.9mg/mL and 2.8mg/mL by BCA method.2. Preparation of monoclonal antibody against DCIn this study, we use DC-BSA to immunized femal BALB/c mice, DC-OVA as coating antigen to peridium ELISA plate. Seventh days after the five immunization, the titer of serum was detected by 1:10000. By using the technology of cell fusion, ELISA and limited dilution, we obtain two monoclonal antigens against DC. They were named 6C9 and 1H8. Two kinds of the ascites were prepared by expanding the two cells and injecting cells into mice abdomen. The two titers were 1:200000 and 1:160000, the protein concentration of the two ascites were 31.6mg/mL and 25.2mg/mL.3. The establishment of ELISA detection methodThe indirect competitive ELISA methods for detection of DC drug residues were established by using purfied 6C9 monoclonal antibody. The best dilution of original package is 1:3000 and the optimal working concentration of 6C9 antibody is 1:32000. Using DC to produce drug competition inhibition curve, the standard curve of a good linear equation for y=0.346x-0.212(R2=0.987), IC50=126.5ng/mL, LOD below lOng/mL. The cross experiment result is that the inhibition of DC monoclonal antibody 6C9 rate is 100%, the cross reaction rate of tetracycline is 26.6%, and the cross reaction rate of oxytetracycline is 14.8%, chlortetracycline has no cross reaction.4. The addition and reclaim test of DC in the porkThe indirect competitive ELISA was used to detect DC residues in the pork. The pork sample was determined after pre-processing. The results show that when the sample solution is diluted 8 times, the matrix interference is disappeared. In the 5-1000ng/mL range, the curve standard equation was:y=0.308x-0.132, R2=0.967, IC50 was 123.6ng/mL, LOD lowed 6ng/mL. When adding 5,200, 1000ng/mL DC to the pork, the recovery rate was between 80% to 98%. |
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