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| 双抗体夹心酶联免疫吸附法检测小麦中禾谷镰孢菌 |
| Detection of Fusarium graminearum in wheat by double-antibody sandwich enzyme-linked immunosorbent assay |
| 投稿时间：2025-02-18 修订日期：2025-05-12 |
| DOI： |
| 中文关键词: 禾谷镰孢菌 单克隆抗体 免疫测定 小麦 霉菌毒素 |
| 英文关键词:Fusarium graminearum Monoclonal antibody Immunoassay Wheat Mycotoxin |
| 基金项目:国家自然科学基金项目(32102089, 32422072, U22A20551) |
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| 中文摘要: |
| 目的 研制禾谷镰孢菌的特异性抗体，开发小麦禾谷镰孢菌污染情况的双抗体夹心酶联免疫吸附测定法(double-antibody sandwich enzyme-linked immunosorbent assay, DAS-ELISA)。方法 采用物理研磨法制备禾谷镰孢菌真菌裂解液抗原，通过杂交瘤技术研制禾谷镰孢菌单克隆抗体(monoclonal antibody, mAb)，对mAb的特异性、亲和力等抗体性能参数进行测定。采用棋盘法探究制备的mAb与多克隆抗体(polyclonal antibody, pAb)最佳组合条件，进而建立DAS-ELISA方法。对方法的灵敏度、重复性进行评价，并应用于自然发病小麦样品的检测。结果 成功制备了禾谷镰孢菌mAb 1C4F3和pAb FG2801。mAb与其他真菌无交叉反应，特异性强。亲和力常数为1.26×107 L/mol，具有较高的亲和力。建立了禾谷镰孢菌DAS-ELISA方法，检测限达1.223 μg/mg，标准曲线范围1.56~100.00 μg/mg。通过对从中国主要小麦产区采集的96份自然发病样本进行检测，验证了该方法的可靠性。发现样品中禾谷镰孢菌的含量与脱氧雪腐镰刀菌烯醇(deoxynivalenol, DON)的含量存在显著的正相关关系(R2=0.8897)，因此DAS-ELISA不仅可用于禾谷镰孢菌的快速检测，还可评估样本中DON的污染情况。结论 本研究开发的DAS-ELISA能够对小麦中的禾谷镰孢菌进行快速定量测定，同时可快速评估样本中DON的污染水平，具有较高的准确性和可靠性，在田间及收获后小麦作物禾谷镰孢菌污染监测方面具有良好的应用潜力。 |
| 英文摘要: |
| Objective To develop specific antibodies against Fusarium graminearum and establish a double - antibody sandwich enzyme - linked immunosorbent assay (DAS - ELISA) for detecting the contamination of Fusarium graminearum in wheat. Methods The fungal lysate antigen of Fusarium graminearum was prepared by physical grinding. Monoclonal antibodies (mAbs) against Fusarium graminearum were developed using hybridoma technology. The performance parameters of mAbs, such as specificity and affinity, were measured. The optimal combination conditions of the prepared mAbs and polyclonal antibodies (pAbs) were explored using the checkerboard method, and then the DAS - ELISA method was established. The sensitivity and repeatability of the method were evaluated, and the method was applied to the detection of naturally infected wheat samples. Results The mAb 1C4F3 and pAb FG2801 against Fusarium graminearum were successfully prepared. The mAb showed no cross - reaction with other fungi, indicating strong specificity. The affinity constant was 1.26×10? L/mol, suggesting high affinity. A DAS - ELISA method for Fusarium graminearum was established, with a detection limit of 1.223 μg/mg and a standard curve range of 1.56 - 100.00 μg/mg. The reliability of the method was verified by detecting 96 naturally infected samples collected from major wheat - producing areas in China. A significant positive correlation was found between the content of Fusarium graminearum and the content of deoxynivalenol (DON) in the samples (R2 = 0.8897). Therefore, the DAS - ELISA can not only be used for the rapid detection of Fusarium graminearum but also for evaluating the DON contamination in the samples. Conclusion The DAS - ELISA developed in this study enables rapid and quantitative determination of Fusarium graminearum in wheat. Simultaneously, it allows for a swift assessment of the DON contamination level in samples. Characterized by high accuracy and reliability, this method holds considerable application potential in monitoring Fusarium graminearum contamination in wheat crops both in the field and post - harvest. |
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