[脱氧雪腐镰刀菌烯醇单克隆抗体的研制]

[Development of the monoclonal antibody to deoxynivalenol].

作者信息

Ji Fang, Chen Zhengxian, Xu Jianhong, Lu Qiongxian, Shi Jianrong

机构信息

Jiangsu Academy of Agriculture Science, Nanjing 210014, China.

出版信息

Wei Sheng Wu Xue Bao. 2008 Jul;48(7):929-34.

PMID:18837372

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 x 10(-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 kappa. Ascites antibodies generated by hybridoma of 3B2 cells were purified. Inhibition rate studies showed that the detection limit of the ELISA was 8 microg/L, with the regression equation of indirect ELISA Y = 0.7331g(x)-0.572, R2=0.9652, IC50 value being 29 ug/L.

CONCLUSION

The MAb can be used to prepare the reagents for analyzing DON residue.

摘要

目的

脱氧雪腐镰刀菌烯醇（DON）是由禾谷镰刀菌产生的一种单端孢霉烯族霉菌毒素，禾谷镰刀菌是引起小麦赤霉病的病原菌。建立一种快速简便的检测DON的方法很有必要。

方法

采用500mg/mL聚乙二醇400进行细胞融合，在HAT（H：次黄嘌呤，A：氨基蝶呤；T：胸腺嘧啶核苷）培养基中进行细胞亚克隆以筛选和有限稀释，制备针对DON的高亲和力单克隆抗体（Mab）。通过酶联免疫吸附测定（ELISA）筛选杂交瘤细胞系对DON的特异性。用与卵清蛋白（OVA）偶联的人工抗原免疫BAL B/C小鼠，取其脾细胞与小鼠骨髓瘤细胞（SP 2/0）融合，获得一株分泌抗DON单克隆抗体（MAb）的杂交瘤细胞系（3B2）。

结果

本实验获得的MAb能与DON特异性反应，除3-乙酰脱氧雪腐镰刀菌烯醇（3-Ac-DON）外，与DON相关化合物无交叉反应，间接ELISA法测定腹水效价高达1×10⁻⁷。单克隆细胞系（3B2）的免疫球蛋白类型和亚类显示其属于IgG1。鉴定该MAb的轻链为κ链。对3B2细胞杂交瘤产生的腹水抗体进行纯化。抑制率研究表明，ELISA法的检测限为8μg/L，间接ELISA回归方程为Y = 0.7331g(x)-0.572，R² = 0.9652，半数抑制浓度（IC50）值为29μg/L。