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首页> 外文期刊>Fish & Shellfish Immunology >Development of a sandwich ELISA for quantifying hepcidin in Rainbow trout
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Development of a sandwich ELISA for quantifying hepcidin in Rainbow trout

机译：建立用于定量虹鳟鱼中铁调素的夹心ELISA的方法

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One of the most widespread antimicrobial peptides (AMPs) in fish is the hepcidins, which have potent, broad-spectrum activity against viruses, bacteria, fungi, and parasites. Moreover, they play the role of central regulation of iron metabolism and their expression is over-regulated by bacterial and viral infections, inflammation and vaccination. Quantification of their expression is an important factor in understanding their function. We therefore generated two polyclonal antibodies using synthetic peptides in order to measure hepcidin expression via sandwich ELISA. The specificity of both antibodies was confirmed by identifying an absence of cross-reactivity with other peptides that have similar pI and with the detection by Western blot of only one 9.6 kDa immunoreactive band corresponding to the hepcidin prepropeptide. The sensitivity of the sandwich ELISA was in the order of 0.005 ng/mu L of hepcidin, which allowed analysis of the presence of the peptide and its variation in different tissues of Oncorhynchus mykiss. With the sandwich ELISA it could be seen that hepcidin expression in rainbow trout challenged with Aeromonas salmonicida was increased twofold over the untreated fish in head kidney samples, in correlation with the increase in the observed transcriptional level in the head kidney cells. These results provide the first evidence for quantifying the presence of active hepcidin and may be a useful indicator of disease susceptibility, providing a new, sensitive tool for rapid screening of population health. (C) 2013 Published by Elsevier Ltd.
机译：铁调素是鱼类中最广泛使用的抗菌肽（AMPs）之一，对病毒，细菌，真菌和寄生虫具有有效的广谱活性。而且，它们起着铁代谢的中央调节作用，并且它们的表达由于细菌和病毒感染，炎症和疫苗接种而被过度调节。量化它们的表达是理解其功能的重要因素。因此，我们使用合成肽生成了两种多克隆抗体，以便通过夹心ELISA测量hepcidin的表达。通过鉴定不存在与具有相似pI的其他肽的交叉反应，并通过Western印迹检测仅对应于hepcidin前原肽的一个9.6 kDa免疫反应带，证实了这两种抗体的特异性。夹心ELISA的灵敏度约为0.005 ng /μL的hepcidin，可以分析该肽的存在及其在Oncorhynchus mykiss的不同组织中的变异。通过夹心ELISA，可以看出，在头肾脏样品中，用鲑鱼气单胞菌攻击的虹鳟鱼中铁调素的表达是未经处理的鱼的两倍，这与头肾脏细胞中观察到的转录水平的增加有关。这些结果为量化活性铁调素的存在提供了第一个证据，并且可能是疾病易感性的有用指标，为快速筛查人群健康提供了新的敏感工具。 （C）2013由Elsevier Ltd.出版

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