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浙江大学学报（农业与生命科学版）
  2012, Vol. 38 Issue (3): 347-354    DOI: 10.3785/j.issn.1008-9209.2012.03.016
Resource & environmental sciences     
Quantitative analysis of cholinesterase from Daphnia magna  by indirect and non-competitive enzyme-linked immunosorbent assay.
LIU Hongcui1, YANG Yanxia2, LI Shaonan1
1. Institute of Pesticide and Environmental Toxicology, Zhejiang University, Hangzhou 310029, China; 2. NO.1 Middle School of Haining, Haining, Zhejiang 314400，China
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Abstract  Polyclonal anti-serum of cholinesterase with dilution ratio of 1∶8000 was obtained by immunizing BALB/c mice with purified cholinesterase （ChE） from Daphnia magna． An indirect and non-competitive enzyme-linked immunosorbent assay (ELISA), which used mice antiDaphnia ChE as antibodies to react with the Daphnia ChE, was established to quantify immunoreactive content of ChE in D. magna. The minimal detection limit of the method was estimated to be 24 ng·mL-1. Specificity of antiserum was high, with cross reaction of <0.25%, <0.25%, 2.13%, 10.19%, 3.88%, 3.56%, 2.81%, 3.93%, 5.00% and 2.75% towards non-ChE marker (5, 10 μg·mL-1), Chironomus kiinensis, Alona sp. , Brachydanio rerio (the brain, the whole body without brain), Bombyx mori, Apis mellifera L., Eisenia foetida, and tadpoles of Xenopus laevis, respectively. It is indicated  that the  established method can  be used to quantify immunoreactive content of ChE in D. magna so as to  measureme accurately  in vivo activity of the ChE in pesticide exposure.

Published: 24 May 2012
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LI Shaonan
LIU Hongcui
YANG Yanxia
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LIU Hongcui, YANG Yanxia, LI Shaonan. Quantitative analysis of cholinesterase from Daphnia magna  by indirect and non-competitive enzyme-linked immunosorbent assay.. , 2012, 38(3): 347-354.

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http://www.zjujournals.com/agr/10.3785/j.issn.1008-9209.2012.03.016     OR     http://www.zjujournals.com/agr/Y2012/V38/I3/347


大型溞胆碱酯酶间接非竞争酶联免疫吸附定量分析法的建立

用达到电泳纯的大型溞（Daphnia magna）胆碱酯酶（cholinesterase, ChE）免疫BALB/c小鼠，得到效价达1∶8000的鼠抗大型溞ChE多克隆抗血清；通过优化抗原抗体反应条件，建立定量分析大型溞ChE含量的间接非竞争酶联免疫吸附法（indirect and non-competitive enzyme-linked immunosorbent assay）。结果表明：此方法检测灵敏度为24 ng·mL-1；经免疫交叉反应鉴定，所获抗血清与5、10 μg·mL-1标准蛋白质和其他生物如花翅摇蚊、尖额溞、斑马鱼（脑、脑除外的整体）、家蚕、意大利蜜蜂、赤子爱胜蚯蚓、非洲爪蟾（蝌蚪）等来源的ChE交叉反应率分别为：<0.25%、<0.25%、2.13%、10.19%、3.88%、3.56%、2.81%、3.93%、5.00%和2.75%，具较高的特异性。说明用本试验得到的抗血清建立的间接非竞争酶联免疫吸附法可用于大型溞体内ChE含量的测定，以利于在农药暴露环境下大型溞体内ChE活性的精确检测。
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