Preparation Of Monoclonal Antibodies Against Ceftraxone And Initially Established Of An ELISA Method

Ceftraxone is the third generation of cephalosporins antibiotics.It often used for the treatment of animal infectious diseases because of its broad antibacterial spectrum and antibacterial ability.However,the abuse of ceftriaxone leads to human and animal diseases of a variety of systems easily,and the most serious disease is allergic reactions and stones.Ceftriaxone is a human antibiotic and it has been listed by the Ministry of Agriculture as a Human medicine prohibited using for Animal breeding process.We can use physical and chemical detection method,microbial detection method and immunological detection method to detect the residual of cephalosporin antibiotic.Physical and chemical detection method requires valuable equipment,highly skilled technical person and complex pretreatment of sample.These restrictions determine the physical and chemical testing method does not suitable for a large number of grass-roots detection of samples.Microbiological detection method takes much time in bacterial culture;the specificity detection is not high;the test results are low accuracy.ELISA and other immunological methods which based on antigen-specific binding.It have strong specificity,high sensitivity,simple method of operation,which suitable for screening a large number of samples.1.Synthesis and identification of complete antigen of CTRXIn this study,we used glutaraldehyde to synthesize complete antigen.The complete antigen was identified by UV spectroscopy and SDS-PAGE Gel electrophoresis.The results showed that the coupling was successful in initial determination.After immunization,cell fusion and ELISA assay,the final proof of two complete antigen preparation was successful.The concentrations of CTRX-BSA and CTRX-OVA were 5.5 mg/mL and 5.0 mg/mL by using BCA method.2.CTRX monoclonal antibody preparationIn this study,we use conventional immunization regimens to immunize BALB/c mice,using the artificial antigen CTRX-BSA and using the CTRX-OVA to detect in ELISA experiment.Seventh days after the five immunization,we collect blood from the tail vein to measure the potency of serum antibody and its drug inhibition.The results showed that the serum titer was 1:16000 or more.We got two hybridoma cell lines who could be secreting monoclonal antibodies againsting CTRX,by cell fusion,ELISA and limited dilution subcloning technology,which named 3E7 and 7A7.We got ascites by injecting cells of hybridomas into mice abdomen,and the ascites were 18 mL.The protein concentrations were 20.93 mg/mL and 21.47 mg/mL respectively by BCA method,and the ascites titers were 32000 and 128000 respectively.1mL 7A7 ascites was purified by HiTrap Protein G HP.We got 5 mL purified 7A7 ascites.The protein concentration was 1.1 mg/mL and the protein recovery was 5.1%.SDS-PAGE electrophoretogram showed that the effect of ascites purification was good.The results of the cross-experiments showed that the CTRX monoclonal antibody had no cross-reactivity with cephalexin,ceftiofur,cefradine,cefotaxime and cefalotin almost,and the inhibition rate for CTRX was 100%.3.The establishment of ELISA detection methodThe CiELISA method for detecting CTRX residues was established by using purified 7A7 ascites.The best dilution concentration 1:3000 and the best working concentration of purified 7A7 antibody is 1:16000.When the CTRX concentration was between 1.72 and 2000 ng/mL,the linearity of the standard curve was good.The linear equation was y=0.2541x-0.0184(R2=0.9875),IC50=108.68 ng/L,LOD=1.72 ng/mL and LOQ=13.12 ng/mL.4.The addition and reclamation test of CTRX in the porkThe addition and reclamation test of CTRX was carried on in the pork.The sample was determined after pre-processing.The experimental results showed that the sample treatment diluted more than 8 times,and it could eliminate the interference of the sample matrix on the whole.The linearity was good in the range of 20?1000 ng/mL.The linear relationship was y=0.4466x-0.4738(R2=0.9869),IC50=150.52 ng/mL,LOD=16.38 ng/mL,LOQ=44.53 ng/mL.When adding 50,400,800 ng/mL CTRX standard solution to do the addition and reclamation test,the detection of pork sample recovery rate was between 81%and 98.7%.