抗脱氧雪腐镰刀菌烯醇单克隆抗体的制备

Development of the Monoclonal Antibody to Deoxynivalenol

摘要:

【目的】小麦赤霉病菌产生的毒素不仅在病害发展过程中具有加重赤霉病的作用，而且污染谷物导致严重的食用安全性问题。由于赤霉病的普遍发生，有必要建立快速、灵敏、有效的毒素检测方法，本试验旨在制备可用于检测被脱氧雪腐镰刀菌烯醇污染的粮谷类特异性单克隆抗体。【方法】本实验首先将脱氧雪腐镰刀菌烯醇（DON）的衍生物3-半琥珀酰-脱氧雪腐镰刀菌烯醇(3-HS-DON-OVA)与卵清蛋白（OVA）采用碳化二亚胺法进行偶联得到人工抗原，以此人工抗原免疫BAL B/C 小鼠，取该鼠脾细胞与SP2/O鼠骨髓瘤细胞融合，经筛选和克隆，得到了1株能稳定分泌DON抗体的单克隆细胞株(3B2)，并制备单克隆抗体腹水。【结果】经检测3B2的抗体类型及亚类均为IgG1，其轻链为k链。腹水通过间接酶联免疫吸附测定效价在1×10-7 以上。该单克隆抗体与脱氧雪腐镰刀菌烯醇特异性结合反应的50%抑制质量浓度为29 mg/L，除与3-acetyldeoxynivalenol（3-Ac-DON）的交叉反应率为78.38%，与其他脱氧雪腐镰刀菌烯醇结构类似物无交叉反应。【结论】本实验所制备的单克隆抗体有较高的灵敏度和特异性，具有较好的应用价值。

Abstract:

[Objective] Deoxynivalenol (DON) is a trichothecene mycotoxin produced by Fusarium graminearum, a pathogen causing Fusarium Head Blight of wheat. It is necessary to establish a rapid and simple assay to detect DON. [Methods] High affinity monoclonal antibodies (Mab) against DON were produced by cell fusion with 500 mg/mL Polyethylene Glycol 400, and cell sub-cloning in HAT (H: hypoxanthine, A: aminopterin; T: thymidine) culture medium for screening and limiting dilution. Hybridoma lines were screened for specificity to DON by Enzyme-linked Immunosorbent Assay (ELISA). One hybridoma cell line (3B2) secreting monoclonal antibody (MAb) against DON was produced by fusing mouse myeloma cells (SP 2/0) with spleen cells from BAL B/C mice which were immunized by the artificial antigen conjugated with Ovalbumin (OVA). [Results] The MAb obtained in this experiment could specifically react with DON without cross-reactivity to DON related compounds except 3-acetyldeoxynivalenol (3-Ac-DON), with the titres of ascitic fluids up to 1× l0-7 by indirect ELISA. Isotype and subclass of the monoclonal cell line (3B2) showed that it belonged to IgG1. The light chain of the MAb was identified to be κ. Ascites antibodies generated by hybridoma of 3B2 cells were purified. Inhibition rate studies showed that the detection limit of the ELISA was 8 mg/L, with the regression equation of indirect ELISA Y=0.733lg(x)-0.572, R2=0.9652, IC50 value being 29ug/L. [Conclusion] The MAb can be used to prepare the reagents for analyzing DON residue.