Monoclonal antibody-based serological methods for detecting Citrus tristeza virus in citrus groves
Monoclonal antibody-based serological methods for detecting Citrus tristeza virus in citrus groves
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DOI：
10.1007/s12250-016-3718-4
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发表时间：
2016-08-01
影响因子：
5.5
通讯作者：
Wu, Jianxiang
中科院分区：
文献类型：
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作者：
Liu, Zhen;Chen, Zhe;Wu, Jianxiang
柑桔tristeza病毒（CTV）是柑桔经济上最重要的病毒之一，危害着全世界的柑桔产业。为建立可靠、有效的CTV血清学检测方法，利用表达载体pET-28a在大肠杆菌BL21 （DE3）中表达CTV主要衣壳蛋白（CP）基因，并通过Ni+-NTA亲和层析纯化CTV。用重组蛋白免疫BALB/c小鼠。通过常规杂交瘤技术获得4株分泌CTV单克隆抗体（mab）的杂交瘤细胞株（14B10、14H11、20D5和20G12）。间接酶联免疫吸附试验（ELISA）测定4种杂交瘤细胞系分泌的含单克隆抗体的腹水滴度为10(-6)~ 10（-7）。Western blots检测结果表明，4种单克隆抗体均能与CTV蛋白特异性反应。利用制备的单克隆抗体，分别建立了dot-ELISA、Tissue print-ELISA和triple antibody sandwich (TAS)-ELISA检测树木苗圃CTV和流行病学研究。建立的dot-ELISA法和TAS-ELISA法分别以1:2560和1:10、240 （w/v, g/mL）的稀释度检测柑桔病叶粗提物中的CTV。组织打印酶联免疫吸附试验（Tissue print-ELISA）对大规模现场样品检测特别有用，主要是因为它简单且缺乏样品制备要求。实地调查发现，CTV在中国重庆市、江西省和浙江省的柑橘树上普遍存在。血清学与RT-PCR检测结果符合率达99.5%以上。制备的CTV单克隆抗体和建立的CTV敏感特异性血清学检测方法对我国CTV的检测和防治具有重要意义。
Citrus tristeza virus (CTV) is one of the most economically important citrus viruses and harms the citrus industry worldwide. To develop reliable and effective serological detection assays of CTV, the major capsid protein (CP) gene of CTV was expressed in Escherichia coli BL21 (DE3) using the expression vector pET-28a and purified through Ni+-NTA affinity chromatography. The recombinant protein was used to immunize BALB/c mice. Four hybridoma cell lines (14B10, 14H11, 20D5, and 20G12) secreting monoclonal antibodies (MAbs) against CTV were obtained through conventional hybridoma technology. The titers of MAb-containing ascitic fluids secreted by the four hybridoma lines ranged from 10(-6) to 10(-7) in indirect enzyme-linked immunosorbent assay (ELISA). Western blots showed that all four MAbs could specifically react with CTV CP. Using the prepared MAbs, dot-ELISA, Tissue print-ELISA, and triple antibody sandwich (TAS)-ELISA were developed to detect CTV in tree nurseries and epidemiological studies. The developed dot-ELISA and TAS-ELISA methods could detect CTV in crude extracts of infected citrus leaves with dilutions of 1:2560 and 1:10, 240 (w/v, g/mL), respectively. Tissue print-ELISA was particularly useful for large-scale field sample detection, mainly owing to its simplicity and lack of sample preparation requirements. The field survey revealed that CTV is prevalent on citrus trees in the Chongqing Municipality, Jiangxi Province, and Zhejiang Province of China. The coincidence rate of serological and RT-PCR test results reached more than 99.5%. The prepared MAbs against CTV and established sensitive and specific serological assays have a significant role in the detection and prevention and control of CTV in our country.