The Development Of A Rapid ELISA Test Kit For CPFX Detection In Aquatic Product

The artificial antigen Ciprofloxacin-bovine serum albumin (CPFX-BSA) and Ciprofloxacin ovalbumin (CPFX-OVA) were synthesized by carbodiimide (EDC) method and isobutyl chloroformate method, respectively. The conjugations were preliminary identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) , then measured the protein concentration by Coomassie brilliant blue determination. The result showed that the protein concentration of CPFX-BSA was 1.24 mg/mL and 0.58 mg/mL for CPFX-OVA. According to the waveforms and the absorption peak changes of the conjugations by UV spectrophotometry, the molecular weight was larger than the carrier protein, which proved CPFX successfully conjugating with BSA and OVA, and the conjugate ratio were 7.2∶1 and 6.4∶1 for CPFX-OVA and CPFX-BSA, respectively. The results of mass spectroscopy showed that the molecular weight of CPFX-BSA was 6.895×104 and 4.586×10⁴ for CPFX-OVA. It was calculated that the conjugate ratio of them were 5.6∶1 and 4.2∶1, respectively. This result was consistent with UV analysis. The mass spectroscopy analysis assured the conjugate ratio and the success in artificial antigens. This experiment took the CPFX-BSA as immunizing antigen and the CPFX-OVA served as coating antigen.The artificial CPFX-BSA was inactivated as antigen to immunizate Balb/c mice. With hybridoma technology, one strain hybridomas secreting McAb against CPFX-BSA named CPFX 5G4 was obtained ultimately. The results showed that the titer of CPFX 5G4 was 1.28×10^-6; the molecules of the Heavy chain and Light chain of the McAb were approximately 50 KD and 25 KD according to SDS-PAGE electrophoresis and the antibodies subtypes was IgG3. The rate of cross reaction between CPFX 5G4 and some QNs was from 8.42% to 17.22%; the rate of cross reaction between CPFX-McAb and enrofloxacin was 62.45%; and between other common drugs without a response. After the optimal conditional experiment, an ic-ELISA standard curve was drawn up and the regression equation was obtained, y = -0.4291x + 1.4802, correlation coefficient R² =0.9928. It was calculated that the IC₅₀ value was 192.44 ng/mL and LOD was 16.29 ng/mL. The range of linearity was from 15.625 to 1000 ng/mL. Took eel blood plasma and muscle as matrix to add CPFX, then detected it using the technique that has developed, the recovery rate was between 60.02% and 110.05%; the average recovery rate were 88.74% and 78.00% respectively, which met the requirements for veterinary drug residue test using ELISA.