Development of a class-specific polyclonal antibody-based indirect competitive ELISA for detecting fluoroquinolone residues in milk

Abstract

Modified 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) method was employed to synthesize the artificial antigen of norfloxacin (NOR), and New Zealand rabbits were used to produce anti-NOR polyclonal antibody (pAb). Based on the checkerboard titration, an indirect competitive enzyme-linked immunosorbent assay (icELISA) standard curve was established. This assay was sensitive and had a working range from 0.12 to 68.40 ng/ml, with the half maximal inhibitory concentration (IC(50)) and limit of detection (LOD) values of 2.7 ng/ml and 0.06 ng/ml, respectively. The produced pAb exhibited high cross-reactivity to fluoroquinolones (FQs) tested, and the IC(50) values to enoxacin, ciprofloxacin, and pefloxacin were 3.1, 3.4, and 4.1 ng/ml, respectively. It also indicated that the concentrations of NaOH and methanol in assay buffer should not be higher than 10% and 30%. When spiked in milk at 5, 20, and 50 ng/ml, the recoveries for NOR, enoxacin, ciprofloxacin, and pefloxacin ranged 90.5%-98.0%, 84.0%-95.2%, 94.0%-106.0%, and 89.5%-100.0%, respectively. The results suggest that this class-specific pAb-based icELISA could be utilized for the primary screening of FQ residues in animal-original products.

Fig. 1

Synthesis process of norfloxacin (NOR) immunogen through 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) method cBSA: cationized bovine serum albumin

Fig. 2

Synthesis procedure for norfloxacin (NOR)-coating antigen through the mixed-anhydride method

Fig. 3

Optimized standard icELISA inhibition curve for NOR NOR-cOVA (2.0 μg/ml) as coating antigen was prepared in CBS (pH 9.6), anti-NOR pAb was diluted 1:16 000 in PBS (pH 7.4), NOR was prepared in PBS containing 20% methanol, and GaRIgG-HRP was diluted 1:1 000 in incubation buffer. Values are expressed as mean±SD (n=3)

Fig. 4

Effects of NaOH (a) and methanol (b) concentrations on the icELISA inhibition curve Insets indicate the fluctuations of A _max/IC₅₀ as a function of solvent concentration. Each value represents the mean of three replicates

Fig. 5

Multiresidue determination icELISA standard curves using class-specific NOR pAb for FQs