Development of a caspase-3 antibody as a tool for detecting apoptosis in cells from rainbow trout (Oncorhynchus mykiss)

Background: Apoptosis is an active cell death process mediated by caspases activation, in which different extrinsic or intrinsic signalling pathways result in direct activation of effector caspases. Caspase-3 is considered to be the most important of the executioner caspases, which cause the morphol...

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Autores principales:	Rojas,Verónica, Guzmán,Fanny, Valenzuela,Cristián, Marshall,Sergio H, Mercado,Luis
Lenguaje:	English
Publicado:	Pontificia Universidad Católica de Valparaíso 2012
Materias:	antibody caspase-3 fish apoptosis Piscirickettsia salmonis
Acceso en línea:	http://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0717-34582012000500012
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spelling	oai:scielo:S0717-345820120005000122012-11-29Development of a caspase-3 antibody as a tool for detecting apoptosis in cells from rainbow trout (Oncorhynchus mykiss)Rojas,VerónicaGuzmán,FannyValenzuela,CristiánMarshall,Sergio HMercado,Luis antibody caspase-3 fish apoptosis Piscirickettsia salmonis Background: Apoptosis is an active cell death process mediated by caspases activation, in which different extrinsic or intrinsic signalling pathways result in direct activation of effector caspases. Caspase-3 is considered to be the most important of the executioner caspases, which cause the morphological and biochemical changes detected in apoptotic cells. Different bacterial and virus pathogens have developed different strategies to survive inside the host and overcome natural protections, one of them is inducing apoptotic death in infected cells. We have demonstrated previously that Piscirickettsia salmonis activates this process in monocytes/macrophages from salmonid RTS11 cell line both by morphological and caspase detection assays; nevertheless, recognition of caspase activation by western blot was impossible since most of the commercially available antibodies for mammalian caspases are not cross-reacting. Results: We have generated a monospecific polyclonal antibody directed to an epitope region of salmonid caspase-3; the selected epitope present high homology with caspase-3 from others teleost species and includes the active site of the enzyme. The peptide was designed using bioinformatics tools and was chemically synthesized using the Fmoc strategy, analysed by RP-HPLC, its molecular weight confirmed by mass spectrometry and its structure analyzed by circular dichroism. The synthetic peptide was immunized and antibodies from ascitic fluid were enriched for immunoglobulins using caprylic acid and then purified by activated affinity columns. The anti-peptide activity of purified antibodies was verified by ELISA, and the ability of the anti-peptide to recognize salmonid caspase-3 activation was demonstrated with the molecule in P. salmonis RTS11 infected cells by western blotting, ELISA and immunocytochemistry. Conclusions: This is the first antibody available for a fish caspase, specifically for trout caspase-3, whose applications were validated by different immunological assays.info:eu-repo/semantics/openAccessPontificia Universidad Católica de ValparaísoElectronic Journal of Biotechnology v.15 n.5 20122012-09-01text/htmlhttp://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0717-34582012000500012en
institution	Scielo Chile
collection	Scielo Chile
language	English
topic	antibody caspase-3 fish apoptosis Piscirickettsia salmonis
spellingShingle	antibody caspase-3 fish apoptosis Piscirickettsia salmonis Rojas,Verónica Guzmán,Fanny Valenzuela,Cristián Marshall,Sergio H Mercado,Luis Development of a caspase-3 antibody as a tool for detecting apoptosis in cells from rainbow trout (Oncorhynchus mykiss)
description	Background: Apoptosis is an active cell death process mediated by caspases activation, in which different extrinsic or intrinsic signalling pathways result in direct activation of effector caspases. Caspase-3 is considered to be the most important of the executioner caspases, which cause the morphological and biochemical changes detected in apoptotic cells. Different bacterial and virus pathogens have developed different strategies to survive inside the host and overcome natural protections, one of them is inducing apoptotic death in infected cells. We have demonstrated previously that Piscirickettsia salmonis activates this process in monocytes/macrophages from salmonid RTS11 cell line both by morphological and caspase detection assays; nevertheless, recognition of caspase activation by western blot was impossible since most of the commercially available antibodies for mammalian caspases are not cross-reacting. Results: We have generated a monospecific polyclonal antibody directed to an epitope region of salmonid caspase-3; the selected epitope present high homology with caspase-3 from others teleost species and includes the active site of the enzyme. The peptide was designed using bioinformatics tools and was chemically synthesized using the Fmoc strategy, analysed by RP-HPLC, its molecular weight confirmed by mass spectrometry and its structure analyzed by circular dichroism. The synthetic peptide was immunized and antibodies from ascitic fluid were enriched for immunoglobulins using caprylic acid and then purified by activated affinity columns. The anti-peptide activity of purified antibodies was verified by ELISA, and the ability of the anti-peptide to recognize salmonid caspase-3 activation was demonstrated with the molecule in P. salmonis RTS11 infected cells by western blotting, ELISA and immunocytochemistry. Conclusions: This is the first antibody available for a fish caspase, specifically for trout caspase-3, whose applications were validated by different immunological assays.
author	Rojas,Verónica Guzmán,Fanny Valenzuela,Cristián Marshall,Sergio H Mercado,Luis
author_facet	Rojas,Verónica Guzmán,Fanny Valenzuela,Cristián Marshall,Sergio H Mercado,Luis
author_sort	Rojas,Verónica
title	Development of a caspase-3 antibody as a tool for detecting apoptosis in cells from rainbow trout (Oncorhynchus mykiss)
title_short	Development of a caspase-3 antibody as a tool for detecting apoptosis in cells from rainbow trout (Oncorhynchus mykiss)
title_full	Development of a caspase-3 antibody as a tool for detecting apoptosis in cells from rainbow trout (Oncorhynchus mykiss)
title_fullStr	Development of a caspase-3 antibody as a tool for detecting apoptosis in cells from rainbow trout (Oncorhynchus mykiss)
title_full_unstemmed	Development of a caspase-3 antibody as a tool for detecting apoptosis in cells from rainbow trout (Oncorhynchus mykiss)
title_sort	development of a caspase-3 antibody as a tool for detecting apoptosis in cells from rainbow trout (oncorhynchus mykiss)
publisher	Pontificia Universidad Católica de Valparaíso
publishDate	2012
url	http://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0717-34582012000500012
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Development of a caspase-3 antibody as a tool for detecting apoptosis in cells from rainbow trout (Oncorhynchus mykiss)

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