Development of a Highly Sensitive and Specific Monoclonal Antibody Based on Indirect Competitive Enzyme-Linked Immunosorbent Assay for the Determination of Zearalenone in Food and Feed Samples

Abstract

Zearalenone (ZEN) contamination in food and feed is prevalent and has severe effects on humans and animals post-consumption. Therefore, a sensitive, specific, rapid, and reliable method for detecting a single residue of ZEN is necessary. This study aimed to establish a highly sensitive and specific ZEN monoclonal antibody (mAb) and an indirect competitive enzyme-linked immunosorbent assay (icELISA) for the detection of ZEN residues in food and feed. The immunogen ZEN-BSA was synthesized via the amino glutaraldehyde (AGA) and amino diazotization (AD) methods and identified using 1H nuclear magnetic resonance (1H NMR), a high-resolution mass spectrometer (HRMS), and an ultraviolet spectrometer (UV). The coating antigens ZEN-OVA were synthesized via the oxime active ester (OAE), formaldehyde (FA), 1,4-butanediol diglycidyl ether (BDE), AGA, and AD methods. These methods were used to screen the best antibody/antigen combination of a heterologous icELISA. Balb/c mice were immunized with a low ZEN-BSA dose at long intervals and multiple sites. Suitable cell fusion mice and positive hybridoma cell lines were screened using a homologous indirect non-competitive ELISA (inELISA) and an icELISA. The ZEN mAbs were prepared by inducing ascites in vivo. The immunological characteristics of ZEN mAbs were then assessed. The standard curves of the icELISA for ZEN were constructed under optimal experimental conditions, and the performance of the icELISA was validated. The two ZEN-BSA immunogens (conjugation ratios, 11.6:1 (AGA) and 9.2:1 (AD)) were successfully synthesized. Four hybridoma cell lines (2B6, 4D9, 1A10, and 4G8) were filtered, of which 2B6 had the best sensitivity and specificity. The mAb 2B6-based icELISA was then developed. The limit of detection (LOD), the 50% inhibitive concentration (IC50), and the linear working range (IC20 to IC80) values of the icELISA were 0.76 μg/L, 8.69 μg/L, and 0.92-82.24 μg/L, respectively. The cross-reactivity (CR) of the icELISA with the other five analogs of ZEN was below 5%. Three samples were spiked with different concentrations of ZEN and detected using the icELISA. The average intra-assay recoveries, inter-assay recoveries, intra-assay coefficients of variations (CVs), and inter-assay CVs were 93.48-99.48%, 94.18-96.13%, 12.55-12.98%, and 12.53-13.58%, respectively. The icELISA was used to detect ZEN in various samples. The results were confirmed using high-performance liquid chromatography/tandem mass spectrometry (HPLC-MS/MS) (correlation coefficient, 0.984). The proposed icELISA was highly sensitive, specific, rapid, and reliable for the detection of ZEN in food and feed samples.

Keywords: highly sensitive and specific monoclonal antibodies; immunoassay; immunogen; indirect competitive enzyme-linked immunosorbent assay (icELISA); zearalenone.

Figure 1

The chemical structure of zearalenone and its homologues: (a) zearalenone (ZEN); (b) alpha-zearalanol (α-ZAL); (c) beta-zearalanol (β-ZAL); (d) zearalanone (ZON); (e) alpha-zearalenol (α-ZEL); (f) beta-zearalenol (β-ZEL).

Figure 2

UV spectra of the ZEN-BSA and ZEN-OVA: (a) UV spectra of ZEN-BSA synthesized via AGA and AD methods; (b) UV spectra of ZEN-OVA synthesized via OAE, FA, BDE, AGA, and AD methods.

Figure 3

The titers and IC50 values for ZEN pAb derived from ZEN-BSA (AGA): (a) the titers for ZEN pAb; (b) the IC50 values for ZEN pAb. The values are the mean of three independent assays (n = 3).

Figure 4

The titers and IC50 values for ZEN pAb derived from ZEN-BSA (AD): (a) the titers for ZEN pAb; (b) the IC50 values for ZEN pAb. The values are the mean of three independent assays (n = 3).

Figure 5

The IC50 and CR values of ZEN pAbs for mouse 5 and mouse 2: (a) the IC50 and CR values of ZEN pAbs for mouse 5; (b) the IC50 and CR values of ZEN pAbs for mouse 2. The data were calculated from triplicate assays.

Figure 6

Characterization of ZEN mAbs obtained in this study: (a) analysis of the subtypes of ZEN mAbs 2B6, 4D9, 1A10, and 4G8; (b) affinity constant (Ka) of ZEN mAbs 2B6, 4D9, 1A10, and 4G8; (c) the titer values of ZEN mAbs 2B6, 4D9, 1A10, and 4G8 at different times; (d) the IC50 values of ZEN mAbs 2B6, 4D9, 1A10, and 4G8 at different times.

Figure 7

Optimization of icELISA experimental conditions and establishment of the icELISA standard curve: (a) selection of different combinations of ZEN mAb and antigen; (b) the effects of methanol contents on the sensitivity of the icELISA (insets indicate the fluctuations of Amax/IC50 as a function of methanol contents); (c) the effects of ionic strengths on the sensitivity of the icELISA (insets indicate the fluctuations of Amax/IC50 as a function of ionic strengths); (d) the effects of pH values on the sensitivity of the icELISA (insets indicate the fluctuations of Amax/IC50 as a function of pH values.

Figure 8

The standard curve of the icELISA for ZEN.

Figure 9

Synthesis route of hapten 5-NH₂-ZEN: (a) zearalenone (ZEN); (b) 5-NO₂-ZEN; (c) 5-NH₂-ZEN.

Figure 10

Synthesis route of immunogen ZEN-BSA via AGA and AD method: (a) zearalenone (ZEN); (b) ZEN-BSA (AGA); (c) ZEN-BSA (AD).

Figure 11

Synthesis routes of coating antigen ZEN-OVA via OAE, FA, BDE, AGA, and AD methods: (a) ZEN-OVA (OAE); (b) ZEN-OVA (FA); (c) ZEN-OVA (BDE); (d) ZEN-OVA (AGA); (e) ZEN-OVA (AD).