Development of a sandwich ELISA for quantifying hepcidin in rainbow trout

Paula A. Santana, Claudio A. Álvarez, Fanny Guzmán, Luis Mercado

Research output: Contribution to journalArticlepeer-review

27 Scopus citations


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 9.6kDa immunoreactive band corresponding to the hepcidin prepropeptide. The sensitivity of the sandwich ELISA was in the order of 0.005ng/μL 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.

Original languageEnglish
Pages (from-to)748-755
Number of pages8
JournalFish and Shellfish Immunology
Issue number3
StatePublished - Sep 2013


  • Antibodies
  • Antimicrobial peptides
  • Fish immunology
  • Immunological methods


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