两种赤潮藻的ELISA检测方法的建立

发布时间：2018-03-14 10:39

本文选题：东海原甲藻　切入点：中肋骨条藻　出处：《中国海洋大学》2014年硕士论文　论文类型：学位论文

【摘要】：近年来，，赤潮的频繁出现对经济和海洋生态系统造成了巨大影响，破坏了海洋生态系统平衡和稳定。解析赤潮暴发的机制，构建检测、预警和防止赤潮暴发的方法和措施成为海洋生态研究的重点。应用ELISA检测方法，利用特定酶与特异性抗体连接的特性，通过测定酶促反应底物的吸光度值可以实现对目的抗原的测定。ELISA检测技术具有较高的灵敏度和重现性，被广泛的应用于微生物检测。在赤潮藻鉴定领域也有所应用。本研究主要针对近年在东海海域引起赤潮暴发现象的东海原甲藻（Prorocentrum donghaiense）和中肋骨条藻（Skeletonema costatum）作为研究对象，制备针对这两种藻细胞蛋白的高特异性的单克隆抗体和多克隆抗体，建立快速、准确、稳定的分析和鉴定赤潮藻的免疫学方法。本研究采用细胞免疫及杂交瘤细胞技术成功的制备了针对东海原甲藻和中肋骨条藻的单克隆抗体和多克隆抗体，建立了针对这两种藻的双抗体ELISA定量检测方法；最低检出限度为1×103cells/mL。用双抗体夹心ELISA定量检测方法与显微镜计数法对单一藻种以及模拟混合藻种进行检测实验，结果发现两者的检测结果高度一致。最后，用本研究建立的抗体定量检测方法对东海赤潮监控海区和嵊泗海水浴场的海水样品进行现场检验，结果表明，本研究建立的针对东海原甲藻和中肋骨条藻的双抗体夹心ELISA检测方法，能够快速完成对这两种赤潮藻的定量检测。本研究建立的双抗体夹心ELISA检测方法可以快速、稳定、准确的特异性检测和识别海水中的东海原甲藻和中肋骨条藻，不会受到其他海洋浮游藻类以及较小悬浮颗粒物的干扰。应用该方法对海水中的东海原甲藻和中肋骨条藻进行检测，可以实现对赤潮暴发的快速检测预报和防治控制。
[Abstract]:In recent years, the frequent occurrence of red tide has a great impact on the economy and marine ecosystem, and has destroyed the balance and stability of marine ecosystem. Methods and measures for early warning and prevention of red tide outbreaks have become the focus of marine ecological research. Using ELISA detection method, using specific enzyme linked to specific antibodies, By measuring the absorbance value of the substrate of enzymatic reaction, the detection of target antigen. Elisa has high sensitivity and reproducibility, and has been widely used in microbial detection, as well as in the field of identification of red tide algae. This study was aimed at Prorocentrum donghaiensee and Skeletonema costatum, which have caused red tide outbreaks in the East China Sea in recent years, and prepared highly specific monoclonal antibodies and polyclonal antibodies against these two kinds of algae cell proteins. To establish a rapid, accurate and stable immunological method for the analysis and identification of red tide algae. In this study, monoclonal antibodies and polyclonal antibodies against Prorocentrum chinensis and P. costatum were successfully prepared by cell immunity and hybridoma cell techniques, and a double antibody ELISA quantitative detection method was established for the detection of these two species of algae. The lowest detection limit is 1 脳 103 cells / mL. The double antibody sandwich ELISA quantitative detection method and microscope counting method are used to detect the single algae species and the simulated mixed algae species. The results show that the detection results are highly consistent. The antibody quantitative detection method established in this study was used to detect the sea water samples in the red tide monitoring area of the East China Sea and Shengsi bathing site. The results showed that, In this study, a double antibody sandwich ELISA method was developed for the detection of Prorocentrum and Ribella costatum in the East China Sea, which can rapidly complete the quantitative detection of these two species of red tide algae. The double antibody sandwich ELISA assay developed in this study can be used to detect and identify Prorocentrum East China Sea and S. costatum in seawater quickly, stably and accurately. This method can be used to detect the Prorocentrum of the East China Sea and the Ribella costatum in the sea water, which can be used to detect and forecast the red tide outbreak quickly and to control the red tide outbreak.
【学位授予单位】：中国海洋大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：X55;X835

【参考文献】