水产养殖病原菌的免疫检测新技术研究

The infection of pathogenic bacteria is an important reason that causes the death of a great number of aquatic animals. The development of a sensitive detection for pathogen is essential in aquaculture. The commonly used florescent antibody and enzyme-linked immunosorbent assay suffer from low sensitivity and the enzyme is easy to be inactive. In time-resolved fluoroimmunoassay (TRFIA), rare-earth chelate is used as a label. Under UV irradiation, the fluorescence lifetime of the chelate is longer than the lifetime of other non-specific fluorescence. Thus, as the fluorescence is measured after a time delay, the background interference could be eliminated, and the detection sensitivity could then be improved. We design this project based on a previously developed work. Aeromonas hydrophila is chosen as an antigen, then the antibody is prepared. The interference of particulate antigen against time-resolved fluorescence is to be focused on. In the meantime, the new soluble rare earth ion chelates with a high fluorescence quantum yield are synthesized and the dissociation enhancement solution is not required. A new determination of A. hydrophila by TRFIA is developed along with biotin - avidin signal amplification technology and monoclonal antibody technology. The sensitivity can be greatly improved. This method will also lay the foundation for TRFIA of other pathogens. TRFIA for aquacultural pathogen is a cross-scientific research between chemical detection methods and aquatic disease prevention. The new detection method can be extended in aquaculture.

病原菌感染会引起水产动物大量死亡，建立灵敏的病原菌检测技术对水产养殖业至关重要。目前常用的荧光抗体法及酶联免疫吸附法存在检测灵敏度不高和酶易失活的缺点，时间分辨荧光免疫检测（TRFIA）采用长寿命的稀土离子螯合物作为标记物，排除了背景荧光干扰，且螯合物比酶稳定，可以解决上述问题。本研究拟在前期工作基础上，以水产养殖重要病原菌嗜水气单胞菌为突破口，制备抗体，并用螯合物标记，重点解决颗粒性抗原对时间分辨荧光的干扰问题。同时针对商业化稀土离子螯合物的不足，合成出荧光量子产率高、不需要离解增强液、易溶的新型螯合物用于TRFIA检测，并结合生物素-亲和素信号放大、单克隆抗体等技术，建立嗜水气单胞菌灵敏、快速、特异的检测方法，也为其它病原菌的TRFIA检测奠定基础。TRFIA用于水产养殖病原菌检测属于化学检测方法与水产病害防治的交叉科学研究，国内外尚未见报道。该方法为水产养殖病原菌检测提供了新的思路。

以水产养殖重要病原菌嗜水气单胞菌为突破口，制备抗体，并用螯合物标记，建立了时间分辨荧光免疫法（TRFIA）检测嗜水气单胞菌。主要完成了以下工作：重点解决了颗粒性抗原对时间分辨荧光的干扰问题，证明了TRFIA发完全可以用于水产养殖致病菌检测，解决两种学科的交叉兼容；充分发挥TRFIA检测灵敏度高的优点，结合生物素-链霉亲和素系统，改进标记方法，检测嗜水气单胞菌灵敏度可达1.0102cfu/mL，远高于荧光抗体法及酶联免疫吸附法（ELISA）；针对商业化稀土离子螯合物的不足，合成了3种新型的稀土离子螯合物，这种螯合物荧光量子产率高、不需要离解增强液、易溶，可用于TRFIA检测；开发新型的酶放大TRFIA技术用于检测嗜水气单胞菌，灵敏度同样高达1.0102cfu/mL；采用双标记TRFIA法同时检测嗜水气单胞菌与产酸克雷伯氏菌，简化了操作步骤，同时可以进行两种病原菌的检测；纳米粒子包埋稀土螯合物，建立了一种基于纳米粒子的TRFIA法检测水产养殖致病菌。该课题属于交叉学科研究，TRFIA为化学中的分析检测方法，病原菌检测属于水产病害防治，研究开拓了TRFIA的应用范围，课题的顺利完成，对于水产养殖病害的早期防治，有效预防和控制病害的发生，减小病害所造成的经济损失，从而降低养殖成本，维护养殖区域水体生态平衡意义重大，更为重要的是为病原菌检测提供了新的思路。