title: Development of a sandwich ELISA for quantifying hepcidin in Rainbow trout.
abstract: 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/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.
authors.affiliation: Grupo de Marcadores Inmunológicos, Laboratorio de Genética e Inmunología Molecular, Instituto de Biología, Pontificia Universidad Católica de Valparaíso, Valparaíso, Chile. paula.santana@ucv.cl
authors.forename: Paula A
authors.initials: PA
authors.lastname: Santana
authors.forename: Claudio A
authors.initials: CA
authors.lastname: Álvarez
authors.forename: Fanny
authors.initials: F
authors.lastname: Guzmán
authors.forename: Luis
authors.initials: L
authors.lastname: Mercado
mesh_terms: D048409:Aeromonas salmonicida; D000818:Animals; D023181:Antimicrobial Cationic Peptides / Q000235:genetics / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods / Q000662:veterinary*; D005393:Fish Diseases / Q000276:immunology / Q000378:metabolism; D005786:Gene Expression Regulation / Q000502:physiology; D016905:Gram-Negative Bacterial Infections / Q000276:immunology / Q000378:metabolism; D060226:Head Kidney / Q000378:metabolism; D064451:Hepcidins / Q000378:metabolism*; D007113:Immunity, Innate / Q000502:physiology; D017686:Oncorhynchus mykiss / Q000378:metabolism*
chemical_list: D023181:Antimicrobial Cationic Peptides; D064451:Hepcidins
keywords: Antibodies; Antimicrobial peptides; Fish immunology; Immunological methods