TY - JOUR
T1 - Development of a sandwich ELISA for quantifying hepcidin in rainbow trout
AU - Santana, Paula A.
AU - Álvarez, Claudio A.
AU - Guzmán, Fanny
AU - Mercado, Luis
N1 - Funding Information:
This work was funded by doctoral thesis grant 24110237 from CONICYT Chile and Regular Project 037.418/2012 from the Dirección de Investigación, Pontificia Universidad Católica de Valparaíso . PS and CA are fellows of Advanced Human Capital Formation of CONICYT, Chile. We thank Ms. Drew Neavin and Mr. James Conaghan for their English review assistance.
PY - 2013/9
Y1 - 2013/9
N2 - 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.
AB - 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.
KW - Antibodies
KW - Antimicrobial peptides
KW - Fish immunology
KW - Immunological methods
UR - http://www.scopus.com/inward/record.url?scp=84882923282&partnerID=8YFLogxK
U2 - 10.1016/j.fsi.2013.06.005
DO - 10.1016/j.fsi.2013.06.005
M3 - Article
C2 - 23791861
AN - SCOPUS:84882923282
SN - 1050-4648
VL - 35
SP - 748
EP - 755
JO - Fish and Shellfish Immunology
JF - Fish and Shellfish Immunology
IS - 3
ER -
