Preparation Of Monoclonal Antibodies Against Lomefloxacin And Initially Establishment Of An ELISA Test Method

Lomefloxacin(LMF)is a kind of fluoroquinolone drug(FQs).It is a synthetic antibacterial agent with highly effective.Because of its broad antibacterial spectrum and strong bactericidal effect,it is commonly used to treat bacteria and mycoplasma infections in livestock and poultry breeding.Lomefloxacin is also a commonly used drug in human clinic,and there are also reports of adverse reactions after taking lomefloxacin.Lomefloxacin residue in animal food will be harmful for human health through food chain,on the other hand,it will cause more and more serious drug resistance.Therefore,the Announcement No.2292 issued by the Ministry of agriculture in 2015 clearly indicates that lomefloxacin and other related preparations are prohibited for animal food.The detection of Lomefloxacin residues is mainly based on some physical and chemical methods,such as ultraviolet spectrophotometry,high performance liquid chromatography,fluorescence spectrophotometry,capillary electrophoresis and so on.Although these physical and chemical methods can be used for quantitative and qualitative detection of drugs,the operation process is more complex and is highly dependent on instruments and equipment,so it is not suitable to detect the large quantities of samples in the field.Therefore,it is very important to establish an immunological method for rapid detection of Lomefloxacin residues.1.Synthesis and identification of complete antigen of LMFIn this experiment,two kinds of immunogen LMF-BSA(A)and LMF-BSA(B)were synthesized by carbodiimide method and mixed anhydride method.The coating antigen LMF-OVA was synthesized by carbodiimide method.After UV scanning and SDS-PAGE analysis,the results indicate that the immunogen and the coating antigen were successfully coupled.The mice were immunized with two kinds of immunogen,and after the third immunization,collect blood and use ELISA to detect the serum titers.The serum antibody titers was greater than 1:128000,which proved that the synthetic antigen was successfully synthesized.The BCA kit showed that LMF-BSA(A),LMF-BSA(B)and LMF-OVA were 5.39mg/mL,4.82mg/mL and 5.05mg/mL.The UV Spectrum analysis showed that the coupling ratio of three kinds of artificial antigens were 6.1:1,2.7:1 and 6.5:1.2.Preparation of LMF monoclonal antibody against LMFBALB/c mice were immunized with two kinds of immunogen LMF-BSA(A)and LMF-BSA(B).Seven days after fifth immunization,collect blood and centrifugation,measure the serum titer and drug inhibition.The mice with better immune effect were selected for cell fusion.By using finite dilution method and CiELISA,two hybridoma cell lines secreting LMF monoclonal antibodies were obtained,named 3H8 and 7G4.The cells were injected into the abdominal cavity of mice to induce ascites,and finally collect the ascites 14mL.After the ascites were Purified,the titer of ascites was more than 1:512000,monoclonal antibodies subclasses were IgG1(light chain was Kappa).The cross reactivity of this McAb to mofloxacin,enroflloxacin,ciprofloxacin,salfloxacin and amoxicillin was very low.3.The establishment of ELISA detection methodAfter purification of ascites,a CiELIS A method for detecting LMF residues was established.The optimal working concentration of the antigen and antibody for the monoclonal antibody was found out,the optimum concentration of the coating antigen was 1:4000,the optimum concentration of the antibody was 132000;the best blocking liquid was 0.5%gelatin.When the LMF concentration was between 100 to 10000 pg/mL,the linear relationship was good.The linear equation was y=0.3671x-0.6172(R2=0.9893),IC50=1.10 ng/mL,LOD=65.11 pg/mL,and LOQ-247.84 pg/mL.4.The addition and reclamation test of LMF in the Fresh milkThe addition recovery experiment of LMF was performed on a fresh milk sample.When the milk sample was diluted 16 times,the matrix interference in the milk sample was basically eliminated,When the LMF concentration was between 200-10000 pg/mL,the linear relationship was good.The linear regression equation was y=0.4284x-0.877(R2 = 0.9923),IC50=1.64 ng/mL,LOD=240.93 pg/mL,and LOQ=416.38 pg/mL.When adding high(500 pg/mL),medium(5000 pg/mL),and low(8000 pg/mL)LMF solution to do the drug addition recovery experiments.After the test,the rate of recovery of lomefloxacin in fresh milk addition was calculated,it was between 85,47%to 104.54%.