Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2012 Jul;13(7):545-54.
doi: 10.1631/jzus.B1200001.

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

Guo-ying Fan  1 , Ruo-song Yang, Jin-qing Jiang, Xin-yao Chang, Jun-jie Chen, Yong-hua Qi, Shi-xiu Wu, Xue-feng Yang
Affiliations

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

Guo-ying Fan et al. J Zhejiang Univ Sci B. 2012 Jul.

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.

PubMed Disclaimer

Figures

Fig. 1
Fig. 1
Synthesis process of norfloxacin (NOR) immunogen through 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) method cBSA: cationized bovine serum albumin
Fig. 2
Fig. 2
Synthesis procedure for norfloxacin (NOR)-coating antigen through the mixed-anhydride method
Fig. 3
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
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
Fig. 5
Multiresidue determination icELISA standard curves using class-specific NOR pAb for FQs
See this image and copyright information in PMC

References

    1. Christodoulou EA, Samanidou VF, Papadoyannis IN. Development of an HPLC multi-residue method for the determination of ten quinolones in bovine liver and porcine kidney according to the European Union Decision 2002/657/EC. J Sep Sci. 2008;31(1):119–127. doi: 10.1002/jssc.200700297. - DOI - PubMed
    1. Delepine B, Hurtaud-Pessel D, Sanders P. Simultaneous determination of six quinolones in pig muscle by liquid chromatography-atmospheric pressure chemical ionization mass spectrometry. Analyst. 1998;123(12):2743–2747. doi: 10.1039/a804911d. - DOI - PubMed
    1. Dufresne G, Fouquet A, Forsyth D, Tittlemier SA. Multiresidue determination of quinolone and fluoroquinolone antibiotics in fish and shrimp by liquid chromatography/tandem mass spectrometry. J AOAC Int. 2007;90(2):604–612. - PubMed
    1. Hassouan MK, Ballesteros O, Zafra A, Vílchez JL, Navalón A. Multiresidue method for simultaneous determination of quinolone antibacterials in pig kidney samples by liquid chromatography with fluorescence detection. J Chromatogr B. 2007;859(2):282–288. doi: 10.1016/j.jchromb.2007.10.002. - DOI - PubMed
    1. Hermo MP, Nemutlu E, Kir S, Barrón D, Barbosa J. Improved determination of quinolones in milk at their MRL levels using LC-UV, LC-FD, LC-MS and LC-MS/MS and validation in line with regulation 2002/657/EC. Anal Chim Acta. 2008;613(1):98–107. doi: 10.1016/j.aca.2008.02.045. - DOI - PubMed

Publication types