应用免疫荧光技术对三种赤潮藻的研究
Study of three red tide algae using immuno-fluorescent technique
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摘要: 为探索和建立免疫学结合荧光技术对赤潮生物的定性、定量检测,制备了3种粒径不同的赤潮藻——赤潮异弯藻(Heterosigma akashiwo)、共生甲藻(Symbiodinium sp)、链状亚历山大藻(Alexandrium catenella)的多克隆抗体,并采用封闭吸附处理以提高抗体的特异性;采用间接酶联免疫法检测了抗体的效价;结果表明未经吸附处理的赤潮异弯藻、共生甲藻的效价分别为1:41 000,1:18 000以上,显示是高亲合力的抗体;链状亚历山大藻抗体的效价达到1:3 500,相比另两种藻的效价较低;吸附处理后的3种多克隆抗体的特异性明显提高,而效价有所下降。3种多克隆抗体与各自藻细胞均有很强的结合力,赤潮异弯藻和共生甲藻的荧光信号均匀分布于细胞表面,链状亚历山大藻细胞的荧光信号在细胞表面分布不均匀,主要集中在腹部横沟内。制备的多克隆抗体只与自身藻细胞结合,与另外2种藻细胞无交叉反应。链状亚历山大藻抗体只与自身藻细胞结合,与塔马亚历山大藻无交叉反应,可作为同属内不同种的鉴别。结合了免疫学和荧光技术,具有特异、灵敏、直观和简便的优点,并且费用低,对于开拓赤潮准确定性、定量检测方法和技术具有重要价值。Abstract: To research and establish the qualitative and quantitative detection of red tide organisms via immunology and fluo rescence technique, polyclonal antibodies against three red tide algae with different size, Heterosigma akashiwo, Symnodinium sp.and Alexandrium catenella were raised.Block affinity treatment was done to enhance the specificity of the antibodies.The titer of three polyclonal antibodies was determined by the indirect enzyme-linked immunoso rbent assay, and three algal cells were detected by immuno-fluorescent technique.The result showed that Heterosigma akashiwo and Symnodinium sp.antibodies without block treatment got titer higher than 1:41 000 and 1:18 000, respectively, an indicating of high affinity antibodies.The titer of Alexandrium catenella antibody reached 1:3 500, relatively lower than the previous two.The specificity of three polyclonal antibodies after affinity treatment was raised remarkably.However, the titer declined.All the polyclonal antibodies showed a high affinity with their own cells.The fluorescent signal of Heterosigma akashiwo and Symnodinium sp.antibodies was conferred to the outer cell surface, while those of Alexandrium catenella was patchy distributed mainly around the excavated cingulum.Polyclonal antibodies were only reacted with their own algae.No cross activity with other two algae was detected.The antibody of Alexandrium catenella reacted its own cells, no cross activity was found with Alexandriumt amarense, which will be applicable in distinguishing species within the genus.Combining immunology with fluorescence technique had the advant ages of specific, sensitive, intuitionistic and simple with low cost.The development of the technique and methods will be prompted for the accurately qualitative and quantitative monitoring of red tide algae.
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