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. 2022 Aug 3;14(8):533.
doi: 10.3390/toxins14080533.

Preparation of Monoclonal Antibody against Deoxynivalenol and Development of Immunoassays

Hoyda Elsir Mokhtar  1 , Aidi Xu  1 , Yang Xu  1 , Mohamed Hassan Fadlalla  1 , Shihua Wang  1
Affiliations

Preparation of Monoclonal Antibody against Deoxynivalenol and Development of Immunoassays

Hoyda Elsir Mokhtar et al. Toxins (Basel). .

Abstract

Fusarium toxins are the largest group of mycotoxins, which contain more than 140 known secondary metabolites of fungi. Deoxynivalenol (DON) is one of the most important compounds of this class due to its high toxicity and its potential to harm mankind and animals and a widespread contaminant of agricultural commodities, such as wheat, corn, barley, oats, bread, and biscuits. Herein, a hybridoma cell 8G2 secreting mAb against DON was produced by fusing the splenocytes with a tumor cell line Sp2/0. The obtained mAb had a high affinity (2.39 × 109 L/mol) to DON. An indirect competitive Enzyme-Linked Immunosorbent Assay (ic-ELISA) showed that the linear range for DON detection was 3.125-25 μg/mL, and the minimum inhibitory concentration (IC50) was 18.125 μg/mL with a limit of detection (LOD) of 7.875 μg/mL. A colloidal gold nanoparticle (AuNP) with 20 nm in diameter was synthesized for on-site detection of DON within 10 min with vLOD of 20 μg/mL. To improve the limit of detection, the gold nanoflower (AuNF) with a larger size (75 nm) was used to develop the AuNF-based strip with vLOD of 6.67 μg/mL. Compared to the vLOD of a convectional AuNP-based strip, the AuNF-based strip was three times lower. Herein, three immunoassay methods (ic-ELISA and AuNP/AuNF-based strips) were successfully developed, and these methods could be applied for the DON detection in agricultural products.

Keywords: ELISA; cell fusion; deoxynivalenol; immunochromatographic strips; monoclonal antibody.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Isotype and chromosome assay: (a) Isotype assay of the hybridoma cells 4A4 and 8G2; (b) chromosome analysis of the hybridoma cells 4A4 and 8G2.
Figure 2
Figure 2
Purification and titer assay of ascites and the purified anti-DON mAb: (a) SDS-PAGE assay of ascites and the purified mAb for 4A4; (b) SDS-PAGE assay of ascites and the purified mAb for 8G2. lane 1: marker; lane 2: ascites; lane 3: purified antibody; Hc = heavy chain; Lc = light chain; (c) titer assay of ascites and purified mAb (4A4 and 8G2).
Figure 2
Figure 2
Purification and titer assay of ascites and the purified anti-DON mAb: (a) SDS-PAGE assay of ascites and the purified mAb for 4A4; (b) SDS-PAGE assay of ascites and the purified mAb for 8G2. lane 1: marker; lane 2: ascites; lane 3: purified antibody; Hc = heavy chain; Lc = light chain; (c) titer assay of ascites and purified mAb (4A4 and 8G2).
Figure 3
Figure 3
Affinity and cross-reactivity analysis of anti-DON mAb: (a) affinity determination of anti-DON mAb from 4A4; (b) affinity determination of anti-DON mAb from 8G2; (c) specificity of anti-DON mAb from 4A4; (d) specificity of anti-DON mAb from 8G2.
Figure 4
Figure 4
Development of ic-ELISA: (a) calibration curve of (B/B0) against DON concentration; (b) standard curve for DON determination by ic-ELISA.
Figure 5
Figure 5
Construction of the AuNP-based strip for determination of DON: (a) AuNP strip model; (b) construction of the AuNP-based strip; (c) specificity of the AuNP-based strip; (d) sensitivity of the developed AuNP strip for DON toxin detection; (e) real samples were determined by the developed AuNP-based strip.
Figure 6
Figure 6
Preparation of the AuNF solution and the AuNF probe: (a) preparation of the AuNF solution; (b) the spectra of AuNF solution; (c) the optimal amount of anti-DON mAb for preparation of AuNF probe; (d) optimal volume of K2CO3 for preparation of the AuNF probe.
Figure 7
Figure 7
Construction and characterization of the AuNF strip: (a) sensitivity of the developed AuNF strip for DON toxin detection; (b) specificity of the AuNF strip; (c) detection of DON in real samples using the AuNF strip.
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