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摘要
This article aimed to prepare monoclonal antibody against ciprofloxacin (CIP mAb) and develop a multidetermination immunoassay for fluoroquinolone (FQs) residues. For this purpose, artificial antigen was synthesized using a modified active ester method and identified by the UV-visible spectra and IR Spectrometer. The splenocytes of the immunized BALB/c mice were fused with NS0 myeloma cells, and noncompetitive and indirect competitive ELISA were employed to screen positive cell clones. Based on checkerboard titration, an icELISA standard curve was established, which specificity, physico-chemical factors and spiking tests in milk were evaluated. The results showed that the artificial antigen was synthesized successfully, and molar ratio of 18:1 for CIP-cBSA conjugate was obtained. A broad-specificity mAb named C3D5H3 was screened out, and the established icELISA curve had a linear range from 0.03 to 95.6 ng/ml (R-2 = 0.9663), with IC50 and LOD values of 0.59 ng/ml and 0.02 ng/ml. Of all the competitive analogues, this assay exhibited a high cross-reactivity to norfloxacin (79.7%), enrofloxacin (66.3%), pefloxacin (57.8%), enoxacin (31.7%), sarafloxacin (23.1%) and ofloxacin (14.9%); slight cross-reactivity to lomefloxacin (9.5%) and marbofloxacin (7.0%). It also indicated that the optimized concentrations of pH and NaCl in dilute solution were 7.4 and 0.1 M, respectively; a 10-fold dilution in milk extracts gave an inhibition curve almost the same as that in PBS buffer. When spiked in milk at 50, 100 and 200 ng/ml, the recoveries for ciprofloxacin, norfloxacin, enrofloxacin and pefloxacin ranged 91.5-96%, 92-100%, 95-102% and 91-94%, respectively. It can be seen that the CIP mAb based multidetermination immunoassay could be utilized for the primary screening of nine FQs residues in animal-original products.
