A double-antibody sandwich ELISA based on the porcine circovirus type 2 (PCV2) propagated in cell culture for antibody detection
Autor(a) principal: | |
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Data de Publicação: | 2016 |
Outros Autores: | , , , , |
Tipo de documento: | Artigo |
Idioma: | eng |
Título da fonte: | Pesquisa Veterinária Brasileira (Online) |
Texto Completo: | http://old.scielo.br/scielo.php?script=sci_arttext&pid=S0100-736X2016001201171 |
Resumo: | ABSTRACT: Few studies have described enzyme-linked immunosorbent assays (ELISAs) for the detection of antibodies against porcine circovirus type 2 (PCV2) based on antigens produced in cell culture. Furthermore, few articles have described viral purification techniques for members of the family Circoviridae. This occurs because circoviruses are difficult to isolate, noncytopathogenic, and produce low viral titres in cell culture. Thus, for overcoming these difficulties in the cultivation of PCV2, this study aimed to develop a double-antibody sandwich ELISA based on the cell culture antigen PCV2b for the quantification of anti-PCV2 antibodies. A 20% and 50% discontinuous sucrose cushion was used for viral purification, which enabled the separation of cell culture proteins in the 20% sucrose cushion and a greater viral concentration in the 50% sucrose cushion. Following isopycnic centrifugation, PCV2 was concentrated in the band with density values from 1.330 to 1.395g/cm3. Viral purification was assessed using SDS-PAGE, indirect ELISA and electron microscopy. The standardised ELISA revealed a strong linear correlation (r= 0.826, p<0.001) when compared with a commercial ELISA kit. The assay exhibited low variability (inter-assay coefficient of variation of 4.24% and intra-assay of 1.80%) and excellent analytical specificity conferred by the capture antibody produced in rabbit. Thus, this ELISA is a rapid, specific and convenient method for the detection of antibodies against PCV2 in studies of experimental and natural infection, and in monitoring the response to vaccination on commercial farms. |
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A double-antibody sandwich ELISA based on the porcine circovirus type 2 (PCV2) propagated in cell culture for antibody detectionEnzyme-linked immunosorbent assayisopycnic centrifugationporcine circovirus type 2sucrose cushionantibody.ABSTRACT: Few studies have described enzyme-linked immunosorbent assays (ELISAs) for the detection of antibodies against porcine circovirus type 2 (PCV2) based on antigens produced in cell culture. Furthermore, few articles have described viral purification techniques for members of the family Circoviridae. This occurs because circoviruses are difficult to isolate, noncytopathogenic, and produce low viral titres in cell culture. Thus, for overcoming these difficulties in the cultivation of PCV2, this study aimed to develop a double-antibody sandwich ELISA based on the cell culture antigen PCV2b for the quantification of anti-PCV2 antibodies. A 20% and 50% discontinuous sucrose cushion was used for viral purification, which enabled the separation of cell culture proteins in the 20% sucrose cushion and a greater viral concentration in the 50% sucrose cushion. Following isopycnic centrifugation, PCV2 was concentrated in the band with density values from 1.330 to 1.395g/cm3. Viral purification was assessed using SDS-PAGE, indirect ELISA and electron microscopy. The standardised ELISA revealed a strong linear correlation (r= 0.826, p<0.001) when compared with a commercial ELISA kit. The assay exhibited low variability (inter-assay coefficient of variation of 4.24% and intra-assay of 1.80%) and excellent analytical specificity conferred by the capture antibody produced in rabbit. Thus, this ELISA is a rapid, specific and convenient method for the detection of antibodies against PCV2 in studies of experimental and natural infection, and in monitoring the response to vaccination on commercial farms.Colégio Brasileiro de Patologia Animal - CBPA2016-12-01info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersiontext/htmlhttp://old.scielo.br/scielo.php?script=sci_arttext&pid=S0100-736X2016001201171Pesquisa Veterinária Brasileira v.36 n.12 2016reponame:Pesquisa Veterinária Brasileira (Online)instname:Colégio Brasileiro de Patologia Animal (CBPA)instacron:EMBRAPA10.1590/s0100-736x2016001200005info:eu-repo/semantics/openAccessCruz,Taís F.Kanashiro,Tatiana M.Castro,Alessandra M.M.G. deBaldin,Cintia M.Richtzenhain,Leonardo J.Araujo Jr,João P.eng2017-03-02T00:00:00Zoai:scielo:S0100-736X2016001201171Revistahttp://www.pvb.com.br/https://old.scielo.br/oai/scielo-oai.phpcolegio@cbpa.org.br||pvb@pvb.com.br0100-736X1678-5150opendoar:2017-03-02T00:00Pesquisa Veterinária Brasileira (Online) - Colégio Brasileiro de Patologia Animal (CBPA)false |
dc.title.none.fl_str_mv |
A double-antibody sandwich ELISA based on the porcine circovirus type 2 (PCV2) propagated in cell culture for antibody detection |
title |
A double-antibody sandwich ELISA based on the porcine circovirus type 2 (PCV2) propagated in cell culture for antibody detection |
spellingShingle |
A double-antibody sandwich ELISA based on the porcine circovirus type 2 (PCV2) propagated in cell culture for antibody detection Cruz,Taís F. Enzyme-linked immunosorbent assay isopycnic centrifugation porcine circovirus type 2 sucrose cushion antibody. |
title_short |
A double-antibody sandwich ELISA based on the porcine circovirus type 2 (PCV2) propagated in cell culture for antibody detection |
title_full |
A double-antibody sandwich ELISA based on the porcine circovirus type 2 (PCV2) propagated in cell culture for antibody detection |
title_fullStr |
A double-antibody sandwich ELISA based on the porcine circovirus type 2 (PCV2) propagated in cell culture for antibody detection |
title_full_unstemmed |
A double-antibody sandwich ELISA based on the porcine circovirus type 2 (PCV2) propagated in cell culture for antibody detection |
title_sort |
A double-antibody sandwich ELISA based on the porcine circovirus type 2 (PCV2) propagated in cell culture for antibody detection |
author |
Cruz,Taís F. |
author_facet |
Cruz,Taís F. Kanashiro,Tatiana M. Castro,Alessandra M.M.G. de Baldin,Cintia M. Richtzenhain,Leonardo J. Araujo Jr,João P. |
author_role |
author |
author2 |
Kanashiro,Tatiana M. Castro,Alessandra M.M.G. de Baldin,Cintia M. Richtzenhain,Leonardo J. Araujo Jr,João P. |
author2_role |
author author author author author |
dc.contributor.author.fl_str_mv |
Cruz,Taís F. Kanashiro,Tatiana M. Castro,Alessandra M.M.G. de Baldin,Cintia M. Richtzenhain,Leonardo J. Araujo Jr,João P. |
dc.subject.por.fl_str_mv |
Enzyme-linked immunosorbent assay isopycnic centrifugation porcine circovirus type 2 sucrose cushion antibody. |
topic |
Enzyme-linked immunosorbent assay isopycnic centrifugation porcine circovirus type 2 sucrose cushion antibody. |
description |
ABSTRACT: Few studies have described enzyme-linked immunosorbent assays (ELISAs) for the detection of antibodies against porcine circovirus type 2 (PCV2) based on antigens produced in cell culture. Furthermore, few articles have described viral purification techniques for members of the family Circoviridae. This occurs because circoviruses are difficult to isolate, noncytopathogenic, and produce low viral titres in cell culture. Thus, for overcoming these difficulties in the cultivation of PCV2, this study aimed to develop a double-antibody sandwich ELISA based on the cell culture antigen PCV2b for the quantification of anti-PCV2 antibodies. A 20% and 50% discontinuous sucrose cushion was used for viral purification, which enabled the separation of cell culture proteins in the 20% sucrose cushion and a greater viral concentration in the 50% sucrose cushion. Following isopycnic centrifugation, PCV2 was concentrated in the band with density values from 1.330 to 1.395g/cm3. Viral purification was assessed using SDS-PAGE, indirect ELISA and electron microscopy. The standardised ELISA revealed a strong linear correlation (r= 0.826, p<0.001) when compared with a commercial ELISA kit. The assay exhibited low variability (inter-assay coefficient of variation of 4.24% and intra-assay of 1.80%) and excellent analytical specificity conferred by the capture antibody produced in rabbit. Thus, this ELISA is a rapid, specific and convenient method for the detection of antibodies against PCV2 in studies of experimental and natural infection, and in monitoring the response to vaccination on commercial farms. |
publishDate |
2016 |
dc.date.none.fl_str_mv |
2016-12-01 |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/article |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
format |
article |
status_str |
publishedVersion |
dc.identifier.uri.fl_str_mv |
http://old.scielo.br/scielo.php?script=sci_arttext&pid=S0100-736X2016001201171 |
url |
http://old.scielo.br/scielo.php?script=sci_arttext&pid=S0100-736X2016001201171 |
dc.language.iso.fl_str_mv |
eng |
language |
eng |
dc.relation.none.fl_str_mv |
10.1590/s0100-736x2016001200005 |
dc.rights.driver.fl_str_mv |
info:eu-repo/semantics/openAccess |
eu_rights_str_mv |
openAccess |
dc.format.none.fl_str_mv |
text/html |
dc.publisher.none.fl_str_mv |
Colégio Brasileiro de Patologia Animal - CBPA |
publisher.none.fl_str_mv |
Colégio Brasileiro de Patologia Animal - CBPA |
dc.source.none.fl_str_mv |
Pesquisa Veterinária Brasileira v.36 n.12 2016 reponame:Pesquisa Veterinária Brasileira (Online) instname:Colégio Brasileiro de Patologia Animal (CBPA) instacron:EMBRAPA |
instname_str |
Colégio Brasileiro de Patologia Animal (CBPA) |
instacron_str |
EMBRAPA |
institution |
EMBRAPA |
reponame_str |
Pesquisa Veterinária Brasileira (Online) |
collection |
Pesquisa Veterinária Brasileira (Online) |
repository.name.fl_str_mv |
Pesquisa Veterinária Brasileira (Online) - Colégio Brasileiro de Patologia Animal (CBPA) |
repository.mail.fl_str_mv |
colegio@cbpa.org.br||pvb@pvb.com.br |
_version_ |
1754122236514533376 |