Development and optimization of a monoclonal antibody-based indirect competitive ELISA for detecting fluoroquinolone residue in milk

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 [R Spectrometer. The splenocytes of the immunized BALB/c mice were fused with NSO myeloma cells, and noncompetitive and indirect competitive ELISA were employed to screen positive cell clones. Based on checkerboard titration, an icELIS A 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 forCIP-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 (R3= 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 ranged91.5-96%, 92-100%, 95-102% and 91-94%, respectively. It can be seen that the CIP mAb based multidetermination immunoassay could be utilized for the primaryscreening of nine FQs residues in animal-original products.