Preparation and Characterization of Monoclonal Antibodies with High Affinity and Broad Class Specificity against Zearalenone and Its Major Metabolites
This study aimed to detect and monitor total Zearalenone (ZEN) and its five homologs (ZENs) in cereals and feed. The monoclonal antibodies (mAbs) with a high affinity and broad class specificity against ZENs were prepared, and the conditions of a heterologous indirect competitive ELISA (icELISA) wer...
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MDPI AG
2021-05-01
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| Series: | Toxins |
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| Online Access: | https://www.mdpi.com/2072-6651/13/6/383 |
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| author | Yanan Wang Xiaofei Wang Haitang Zhang Hanna Fotina Jinqing Jiang |
| author_facet | Yanan Wang Xiaofei Wang Haitang Zhang Hanna Fotina Jinqing Jiang |
| author_sort | Yanan Wang |
| collection | DOAJ |
| description | This study aimed to detect and monitor total Zearalenone (ZEN) and its five homologs (ZENs) in cereals and feed. The monoclonal antibodies (mAbs) with a high affinity and broad class specificity against ZENs were prepared, and the conditions of a heterologous indirect competitive ELISA (icELISA) were preliminarily optimized based on the ZEN mAbs. The immunogen ZEN-BSA was synthesized using the oxime active ester method (OAE) and identified using infrared (IR) and ultraviolet (UV). The coating antigen ZEN-OVA was obtained via the 1,4-butanediol diglycidyl ether method (BDE). Balb/c mice were immunized using a high ZEN-BSA dose with long intervals and at multiple sites. A heterologous indirect non-competitive ELISA (inELISA) and an icELISA were used to screen the suitable cell fusion mice and positive hybridoma cell lines. The ZEN mAbs were prepared by inducing ascites in vivo. The standard curve was established, and the sensitivity and specificity of the ZEN mAbs were determined under the optimized icELISA conditions. ZEN-BSA was successfully synthesized at a conjugation ratio of 17.2:1 (ZEN: BSA). Three hybridoma cell lines, 2D7, 3C2, and 4A10, were filtered, and their mAbs corresponded to an IgG1 isotype with a κ light chain. The mAbs titers were between (2.56 to 5.12) × 10<sup>2</sup> in supernatants and (1.28 to 5.12) × 10<sup>5</sup> in the ascites. Besides, the 50% inhibitive concentration (IC50) values were from 18.65 to 31.92 μg/L in the supernatants and 18.12 to 31.46 μg/L in the ascites. The affinity constant (<i>Ka</i>) of all of the mAbs was between 4.15 × 10<sup>9</sup> and 6.54 × 10<sup>9</sup> L/mol. The IC50 values of mAb 2D7 for ZEN, α-ZEL, β-ZEL, α-ZAL, β-ZAL and ZAN were 17.23, 16.71, 18.27, 16.39, 20.36 and 15.01 μg/L, and their cross-reactivities (CRs, %) were 100%, 103.11%, 94.31%, 105.13%, 84.63%, and 114.79%, respectively, under the optimized icELISA conditions. The limit of detection (LOD) for ZEN was 0.64 μg/L, and its linear working range was between 1.03 and 288.55 μg/L. The mAbs preparation and the optimization of icELISA conditions promote the potential development of a rapid test ELISA kit, providing an alternative method for detecting ZEN and its homologs in cereals and feed. |
| first_indexed | 2024-03-10T11:00:27Z |
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| institution | Directory Open Access Journal |
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| last_indexed | 2024-03-10T11:00:27Z |
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| spelling | doaj.art-633bc83d4a9e4150bc2b2aba86b53efa2023-11-21T21:33:28ZengMDPI AGToxins2072-66512021-05-0113638310.3390/toxins13060383Preparation and Characterization of Monoclonal Antibodies with High Affinity and Broad Class Specificity against Zearalenone and Its Major MetabolitesYanan Wang0Xiaofei Wang1Haitang Zhang2Hanna Fotina3Jinqing Jiang4College of Animal Science and Veterinary Medicine, Henan Institute of Science and Technology, Xinxiang 453003, ChinaXinke College, Henan Institute of Science and Technology, Xinxiang 453003, ChinaCollege of Animal Science and Veterinary Medicine, Henan Institute of Science and Technology, Xinxiang 453003, ChinaFaculty of Veterinary Medicine, Sumy National Agrarian University, 40021 Sumy, UkraineCollege of Animal Science and Veterinary Medicine, Henan Institute of Science and Technology, Xinxiang 453003, ChinaThis study aimed to detect and monitor total Zearalenone (ZEN) and its five homologs (ZENs) in cereals and feed. The monoclonal antibodies (mAbs) with a high affinity and broad class specificity against ZENs were prepared, and the conditions of a heterologous indirect competitive ELISA (icELISA) were preliminarily optimized based on the ZEN mAbs. The immunogen ZEN-BSA was synthesized using the oxime active ester method (OAE) and identified using infrared (IR) and ultraviolet (UV). The coating antigen ZEN-OVA was obtained via the 1,4-butanediol diglycidyl ether method (BDE). Balb/c mice were immunized using a high ZEN-BSA dose with long intervals and at multiple sites. A heterologous indirect non-competitive ELISA (inELISA) and an icELISA were used to screen the suitable cell fusion mice and positive hybridoma cell lines. The ZEN mAbs were prepared by inducing ascites in vivo. The standard curve was established, and the sensitivity and specificity of the ZEN mAbs were determined under the optimized icELISA conditions. ZEN-BSA was successfully synthesized at a conjugation ratio of 17.2:1 (ZEN: BSA). Three hybridoma cell lines, 2D7, 3C2, and 4A10, were filtered, and their mAbs corresponded to an IgG1 isotype with a κ light chain. The mAbs titers were between (2.56 to 5.12) × 10<sup>2</sup> in supernatants and (1.28 to 5.12) × 10<sup>5</sup> in the ascites. Besides, the 50% inhibitive concentration (IC50) values were from 18.65 to 31.92 μg/L in the supernatants and 18.12 to 31.46 μg/L in the ascites. The affinity constant (<i>Ka</i>) of all of the mAbs was between 4.15 × 10<sup>9</sup> and 6.54 × 10<sup>9</sup> L/mol. The IC50 values of mAb 2D7 for ZEN, α-ZEL, β-ZEL, α-ZAL, β-ZAL and ZAN were 17.23, 16.71, 18.27, 16.39, 20.36 and 15.01 μg/L, and their cross-reactivities (CRs, %) were 100%, 103.11%, 94.31%, 105.13%, 84.63%, and 114.79%, respectively, under the optimized icELISA conditions. The limit of detection (LOD) for ZEN was 0.64 μg/L, and its linear working range was between 1.03 and 288.55 μg/L. The mAbs preparation and the optimization of icELISA conditions promote the potential development of a rapid test ELISA kit, providing an alternative method for detecting ZEN and its homologs in cereals and feed.https://www.mdpi.com/2072-6651/13/6/383zearalenoneimmunogenmonoclonal antibodieshigh affinity and broad class specificityicELISAimmunoassay |
| spellingShingle | Yanan Wang Xiaofei Wang Haitang Zhang Hanna Fotina Jinqing Jiang Preparation and Characterization of Monoclonal Antibodies with High Affinity and Broad Class Specificity against Zearalenone and Its Major Metabolites Toxins zearalenone immunogen monoclonal antibodies high affinity and broad class specificity icELISA immunoassay |
| title | Preparation and Characterization of Monoclonal Antibodies with High Affinity and Broad Class Specificity against Zearalenone and Its Major Metabolites |
| title_full | Preparation and Characterization of Monoclonal Antibodies with High Affinity and Broad Class Specificity against Zearalenone and Its Major Metabolites |
| title_fullStr | Preparation and Characterization of Monoclonal Antibodies with High Affinity and Broad Class Specificity against Zearalenone and Its Major Metabolites |
| title_full_unstemmed | Preparation and Characterization of Monoclonal Antibodies with High Affinity and Broad Class Specificity against Zearalenone and Its Major Metabolites |
| title_short | Preparation and Characterization of Monoclonal Antibodies with High Affinity and Broad Class Specificity against Zearalenone and Its Major Metabolites |
| title_sort | preparation and characterization of monoclonal antibodies with high affinity and broad class specificity against zearalenone and its major metabolites |
| topic | zearalenone immunogen monoclonal antibodies high affinity and broad class specificity icELISA immunoassay |
| url | https://www.mdpi.com/2072-6651/13/6/383 |
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