Preparation Of Specific Antibody Against Ciprofloxacin And Establishing ELISA

Ciprofloxacin (CIP) belongs to the second-generation fluoroquinolone antibiotic, which has a broad spectrum of antimicrobial activity, it is widely used to treat veterinary diseases in animals intended for human consumption and to treat digestive infections in farming industries as feed additives.The wide spread use of antibiotics in food-producing animals has caused public concern due to the transfer of antibiotic-resistant bacteria to man, leading to the antibiotics treatment useless in common human infections. In many countries, withdrawal period for ciprofloxacin and maximum residue limits (MRLs) for the sum of enrofloxacin and ciprofloxacin have been set for all food producing animals, so powerful and quick analytical methods for surveillance of possible residues of CIP are mandatory.ELISA is a highly specific method for screening biological specimens and has been successfully developed as an alternative to the conventional microbiological or chemical methods for detecting veterinary drug residues. Objective to this study is to prepare CIP specific antibody and develop CIP indirect inhibition ELISA kit to screen CIP residue in food of animal origin, maintaining a safe food supply for export and domestic trade , then obtain better economic and social benefits .Antibody prepration is the critical step of ELISA, since CIP alone is not immunogenic, its conjugation to a carrier protein is the most important step of the project. Several papers have reported the development of the immunoassay for the determination of CIP with different sensitivity, the immunogens were prepared by directly conjugating the carboxyl group of CIP to a carrier protein's amino-group, here we described a more effective method to construct cationized protein using EDA as linker, then conjugateed this cationized carrier to CIP, molar ratio was 20 to 1 as cip to carrier, specific antibody with IC50 at 1ng/mL was obtained from rabbit NO. 2, then we furder optimized the carrier protein, choosing HMD as linker to cationized BSA with a longer space arm ,with the first immunogen as control, to immunize BALB/C with the two immunogen and compare immunological effect, it turned out that the latter immugen could ilicit more specific antibody, which indicated that spacer arm incorpated in immunogen hada great effect on antiserum specifity.After the immunological evaluations were investigated through ELISA, we choose NO.2 rabbit antiserum to develop CIP indirect inhibition ELISA , after the optimal conditional experiment, regression equation of the standard curve was obtained, y=-13.139x+51.3, Correlation coefficient R2=0.998, LOD was 0.01ng/mL, range of linearity was from 0.01 to 10 ng/mL,then we choose chicken muscle,chicken liver and egg as matrix to add CIP ,then detect it using the technique that has developed, recovery rate were 76.95%,79.38% and 79.80% respectively, which meets the requirements for veterinary drug residue test using ELISA.