J Vet Sci.  2012 Jun;13(2):119-125. 10.4142/jvs.2012.13.2.119.

Production of a highly group-specific monoclonal antibody against zearalenone and its application in an enzyme-linked immunosorbent assay

• Cha SH
• Kim SH
• Bischoff K
• Kim HJ
• Son SW
• Kang HG

• KMID: 1376185
• DOI: http://doi.org/10.4142/jvs.2012.13.2.119

Abstract

A monoclonal antibody (mAb) against zearalenone (ZEN) was produced using ZEN-carboxymethoxylamine and -BSA conjugates. Antibody produced by one clone showing a very high binding ability was selected and found to have a higher affinity for ZEN compared to a commerciall ZEN antibody. We developed two direct competitive ELISA systems using the selected antibody (ZEN-coated and anti-ZEN antibody-coated ELISA). Quantitative ranges for the anti-ZEN antibody-coated ELISA and ZEN-coated ELISA were from 25 to 750 ppb and from 12.5 to 100 ppb, respectively. The detection limit of both methods as measured with standard solutions was 10 ppb. The intra-plate and inter-well variation of both ELISAs were less than 10%. The IC50 values for alpha-zearalenol, beta-zearalenol, alpha-zearalanol, and beta-zearalanol compared to ZEN were 108.1, 119.3, 114.1, and 130.3% for the ZEN-coated ELISA. These values were 100.7, 120.7, 121.6, and 151.6% for the anti-ZEN antibody-coated ELISA. According to the anti-ZEN antibody-coated ELISA, the average recovery rates of ZEN from spiked animal feed containing 150 to 600 ng/mL of ZEN ranged from 106.07 to 123.00% with 0.93 to 2.28% coefficients of variation. Our results demonstrate that the mAb developed in this study could be used to simultaneously screen for ZEN and its metabolites in feed.

Keyword

ELISA; group specific; monoclonal antibody; zearalenone

MeSH Terms

Aminooxyacetic Acid/*chemistry
Animals
Antibodies, Monoclonal/*immunology
Enzyme-Linked Immunosorbent Assay/*methods
Female
Inhibitory Concentration 50
Mice
Mice, Inbred BALB C
Reproducibility of Results
Serum Albumin, Bovine/chemistry
Zearalenone/*immunology

Figure

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  Fig. 1 High-performance liquid chromatography chromatogram and UV scanning spectrum of zearalenone (ZEN) (A) and ZEN-oxime (B). The UV spectra of ZEN-carboxymethoxylamine contained three maxima at 236, 271, and 302 nm. This was similar to the spectra of ZEN alone, which had maxima at 235, 272, and 312 nm.

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  Fig. 2 Comparison of affinity to ZEN by anti-ZEN monoclonal antibody (mAb) produced in the current study or commercially available anti-ZEN mAb. C: commercial mAb, kk: kk-ZEN clone. Each point is the mean of three wells.

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  Fig. 3 Standard curves for ZEN for the ZEN-coated ELISA (A) or ZEN antibody-coated ELISA (B). Logit indicate the ratio of "B" and "B0". "B" and "B0" means the absorbance at 450 nm in presence or absence of ZEN, respectively. Each point represents the mean of three wells.

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  Fig. 4 Effect of methanol on the standard curves for the ZENcoated ELISA (A) and ZEN antibody-coated ELISA (B) for ZEN detection in a PBS solution. "B" and "B0" indicate the absorbance at 450 nm in the presence or absence of ZEN, respectively. Each point represents the mean of three wells.

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