Preparation Of Monoclonal Antibodies Against Ciprofloxacin And The Establishment Of ELISA Method

The ciprofloxacin belongs to the fluoroquinolone(FQs) group.It is a kind of antibacterial agent with efficiently broad-spectrum. It has the advantage of rapid drug absorption, bioavailability, widely distributed in vivo, slow drug resistance and no cross-resistance with other antibiotics. So it was widely used to treat veterinary diseases in animals intended for human consumption and to treat digestive infections in farming industries as feed additives. However, as ciprofloxacin was used as a human and animal drugs, the residues of ciprofloxacin is harmful to human health through the food chain and it is not conducive for ciprofloxacin used in the treatment of human disease. U.S, EU, China and more other countries have made a banned or restricted regulation on the limited ciprofloxacin residues in Animal Derived Food. In order to monitor ciprofloxacin residues in livestock products, the establishment of a powerful and quick detection method has great practical significance.With the advantage of highly specific and sensitive, ELISA was widely used to detect veterinary drug residues. To establish the ELISA method for detecting CIP, this research synthesized complete antigen through chemical method, developed high-grade antibodies against ciprofloxacin using the monoclonal antibody technology. The addition and reclamation test of samples proved that the established CiELISA method for detecting the residue of CIP was stable and reliable.1. Synthesis and identification of CIP complete antigensCIP was conjugated with BSA and OVA via NHS ester and mixed anhydride method. And the complete antigen CIP-BSA and CIP-OVA were prepared. The conjugates were preliminary demonstrated to be successful by UV scanning.2. The preparation of the monoclonal antibody against CIPCIP-BSA which was synthesized via NHS ester method was used to immunize BALB/c mouse while CIP-OVA was used for detection. The titer of blood serum collected after the fifth immunization reached1:6,000. Then a hybridoma cell line named6E4which could excrete anti-ciprofloxacin monoclonal antibody steadily was gained by hybridoma technique. Hybridoma6E4excreting anti-ciprofloxacin monoclonal antibody (McAb) was used to inject into mice (ICR) abdominal cavity. The results showed that the protein concentration of McAb was26.5mg/mL, the titer of the McAb was1:24,000.3. The establishment of ELISA methodThrough the establishment of indirect competitive inhibition ELISA, the best concentration of ascites was gained, which is1:20,0O0.CIP standard curve was prepared by6E4ascites, with the linear equation: Y=0.5611x-0.0748(R2=0.9904), IC5010.58ng/mL, LOD was lower than2ng/mL, LOQ5.32ng/mL. The cross reaction experiment showed that the ascites of6E4had good specificity to CIP. And the cross-reactivity with enrofloxacin、 norfloxacin and ofloxacin was34.6%、68.4%and13.4%,the ascites had no cross-reactivity with other non-fluoroquinolone antibiotics.4. The addition and reclamation test of CIPThis method was initially applied to the addition and reclamation test of the chicken liver samples. The intervention disappeared when the extraction of liver sample was diluted1.6times. In the range from4to25ng/mL, the linear equation was Y=0.6515x-0.2937, R2=0.9918, IC50was16.53ng/g, LOD was4.01ng/g. When adding5、15and25ng/mL CIP to the chicken liver, the recovery ratio was between77.8%～97.34%. The intraassay coefficient of variation was between4.63%～16.74%, and the interassay coefficient of variation was between8.57%～10.39%.