Establishment of Indirect Competitive ELISA Method for Detecting Amanitin in Mushroom
Objective: This study aims to establish an indirect competitive enzyme-linked immunosorbent assay for the detection of amanitin (AMA) in mushroom. Methods: In this study, the hapten was obtained by introducing 6-aminocaproic acid into the carboxyl position of the α-amanotropin molecule through EDC/N...
| Main Authors: | , , , |
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| Format: | Article |
| Language: | zho |
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The editorial department of Science and Technology of Food Industry
2022-03-01
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| Series: | Shipin gongye ke-ji |
| Subjects: | |
| Online Access: | http://www.spgykj.com/cn/article/doi/10.13386/j.issn1002-0306.2021060279 |
| _version_ | 1811307651678601216 |
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| author | Hebing LIU Yu QIN Weiwei XING Licai MA |
| author_facet | Hebing LIU Yu QIN Weiwei XING Licai MA |
| author_sort | Hebing LIU |
| collection | DOAJ |
| description | Objective: This study aims to establish an indirect competitive enzyme-linked immunosorbent assay for the detection of amanitin (AMA) in mushroom. Methods: In this study, the hapten was obtained by introducing 6-aminocaproic acid into the carboxyl position of the α-amanotropin molecule through EDC/NHS, and further coupled with different carrier proteins to prepare immunogens and coating antigen. Afterwards, Balb/c mice were immunized with the immunogen to prepare monoclonal antibodies. Based on the obtained antibody, an indirect competitive ELISA method for the detection of amanitin in mushroom was established by optimizing the working concentrations of coating antigen and antibody, coating conditions, blocking conditions, working concentrations of enzyme labeled secondary antibody and incubation time. Finally, the sensitivity, recovery rate, intra batch and inter batch variation of the established method were evaluated. Results: The molecular weight of the synthesized hapten in this study was 1033.12, and the coupling ratio of the immunogen identified by MALDI-TOF was about 10.03. Based on hybridoma technology, the IC50 of the mouse monoclonal antibody 13H4 was 1.91 μg/L. Based on the obtained monoclonal antibody, the detection limit of the established ic-ELISA method for amanitin in mushroom was 0.88 μg/kg, the recovery rate was 85.66%~113.05%, the intra-assay coefficient of variation was 5.35%~9.54%, and the inter-assay coefficient of variation was less than 15%. Conclusion: The ic-ELISA method established in this study had high accuracy, precision, sensitivity, and stable performance. It provided a simple, reliable and rapid detection method for the analysis of the toxicogen of sudden mushroom poisoning. |
| first_indexed | 2024-04-13T09:08:12Z |
| format | Article |
| id | doaj.art-9c8d6e72839f45ffbfb537947a6cbd87 |
| institution | Directory Open Access Journal |
| issn | 1002-0306 |
| language | zho |
| last_indexed | 2024-04-13T09:08:12Z |
| publishDate | 2022-03-01 |
| publisher | The editorial department of Science and Technology of Food Industry |
| record_format | Article |
| series | Shipin gongye ke-ji |
| spelling | doaj.art-9c8d6e72839f45ffbfb537947a6cbd872022-12-22T02:52:56ZzhoThe editorial department of Science and Technology of Food IndustryShipin gongye ke-ji1002-03062022-03-0143529430110.13386/j.issn1002-0306.20210602792021060279-5Establishment of Indirect Competitive ELISA Method for Detecting Amanitin in MushroomHebing LIU0Yu QIN1Weiwei XING2Licai MA3Beijing WDWK Biotechnology Co., Ltd., Beijing 100095, ChinaBeijing WDWK Biotechnology Co., Ltd., Beijing 100095, ChinaBeijing WDWK Biotechnology Co., Ltd., Beijing 100095, ChinaBeijing WDWK Biotechnology Co., Ltd., Beijing 100095, ChinaObjective: This study aims to establish an indirect competitive enzyme-linked immunosorbent assay for the detection of amanitin (AMA) in mushroom. Methods: In this study, the hapten was obtained by introducing 6-aminocaproic acid into the carboxyl position of the α-amanotropin molecule through EDC/NHS, and further coupled with different carrier proteins to prepare immunogens and coating antigen. Afterwards, Balb/c mice were immunized with the immunogen to prepare monoclonal antibodies. Based on the obtained antibody, an indirect competitive ELISA method for the detection of amanitin in mushroom was established by optimizing the working concentrations of coating antigen and antibody, coating conditions, blocking conditions, working concentrations of enzyme labeled secondary antibody and incubation time. Finally, the sensitivity, recovery rate, intra batch and inter batch variation of the established method were evaluated. Results: The molecular weight of the synthesized hapten in this study was 1033.12, and the coupling ratio of the immunogen identified by MALDI-TOF was about 10.03. Based on hybridoma technology, the IC50 of the mouse monoclonal antibody 13H4 was 1.91 μg/L. Based on the obtained monoclonal antibody, the detection limit of the established ic-ELISA method for amanitin in mushroom was 0.88 μg/kg, the recovery rate was 85.66%~113.05%, the intra-assay coefficient of variation was 5.35%~9.54%, and the inter-assay coefficient of variation was less than 15%. Conclusion: The ic-ELISA method established in this study had high accuracy, precision, sensitivity, and stable performance. It provided a simple, reliable and rapid detection method for the analysis of the toxicogen of sudden mushroom poisoning.http://www.spgykj.com/cn/article/doi/10.13386/j.issn1002-0306.2021060279mushroomamanitinic-elisa |
| spellingShingle | Hebing LIU Yu QIN Weiwei XING Licai MA Establishment of Indirect Competitive ELISA Method for Detecting Amanitin in Mushroom Shipin gongye ke-ji mushroom amanitin ic-elisa |
| title | Establishment of Indirect Competitive ELISA Method for Detecting Amanitin in Mushroom |
| title_full | Establishment of Indirect Competitive ELISA Method for Detecting Amanitin in Mushroom |
| title_fullStr | Establishment of Indirect Competitive ELISA Method for Detecting Amanitin in Mushroom |
| title_full_unstemmed | Establishment of Indirect Competitive ELISA Method for Detecting Amanitin in Mushroom |
| title_short | Establishment of Indirect Competitive ELISA Method for Detecting Amanitin in Mushroom |
| title_sort | establishment of indirect competitive elisa method for detecting amanitin in mushroom |
| topic | mushroom amanitin ic-elisa |
| url | http://www.spgykj.com/cn/article/doi/10.13386/j.issn1002-0306.2021060279 |
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