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首页> 外文期刊>The Journal of toxicological sciences >Determination of metallothionein-3 by a competitive enzyme-linked immunosorbent assay in experimental animals
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Determination of metallothionein-3 by a competitive enzyme-linked immunosorbent assay in experimental animals

机译：用竞争性酶联免疫吸附法测定实验动物中的金属硫蛋白-3

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An easy and specific enzyme-linked immunoassay (ELISA) for the determination of metallothinein-3 (MT-3) in experimental animals for the research of heavy metal and chemical toxicity has not been reported yet. Therefore, we have developed a competitive ELISA, using a specific monoclonal antibody raised against human recombinant MT-3 (rMT-3). The epitope mapping of the antibody was conducted using mouse, rat, and human MT-3s and peptide fragments of human MT-3. MT-1/2, MT-3 knock-out (KO) mice and human brain and liver were used for the evaluation of the ELISA. A pretreatment method of the tissue homogenates was also examined. The antibody used for the ELISA had the same cross-reactivity with MT-3 in humans and experimental animals. The human MT- 3 NH2 terminal peptide (Fr. 1-17) was the demonstrated epitope of this antibody. The reactivity of this ELISA in brain homogenate of MT-3 KO mouse was significantly low compared with the wild type and MT-1/2 KO mice. The lowest detection limit of the ELISA was 10 ng/ml and over 80% of the spiked rMT-3 was recovered in the brain homogenate. The assay linearity was intact with a 5-fold dilution in the brain homogenate. The inter- and intra-assay CV was 6.5%, respectively. An effective pretreatment procedure of the tissue homogenate was also established for this MT-3 ELISA. In conclusion, this competitive ELISA is an easy and specific method for measuring the brain MT-3 level in experimental animals.
机译：尚未报道一种用于测定实验动物中重金属和化学毒性的金属硫蛋白-3（MT-3）的简便，特异性的酶联免疫法（ELISA）。因此，我们开发了一种竞争性ELISA，使用针对人类重组MT-3（rMT-3）的特异性单克隆抗体。使用小鼠，大鼠和人MT-3和人MT-3的肽片段进行抗体的表位作图。使用MT-1 / 2，MT-3敲除（KO）小鼠以及人脑和肝脏进行ELISA评估。还检查了组织匀浆的预处理方法。 ELISA中使用的抗体在人和实验动物中与MT-3具有相同的交叉反应性。人MT-3 NH 2 末端肽（Fr. 1-17）是该抗体的表位。与野生型和MT-1 / 2 KO小鼠相比，该ELISA在MT-3 KO小鼠脑匀浆中的反应性明显较低。 ELISA的最低检测限为10 ng / ml，在脑匀浆中回收了超过80％的加标rMT-3。在大脑匀浆中稀释5倍后，测定线性保持不变。批间和批内CV分别为6.5％。还为该MT-3 ELISA建立了组织匀浆的有效预处理程序。总之，这种竞争性ELISA是一种用于测量实验动物脑MT-3水平的简便且特定的方法。

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