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: | Repositório Institucional da UNESP |
Texto Completo: | http://dx.doi.org/10.1590/S0100-736X2016001200005 http://hdl.handle.net/11449/174314 |
Resumo: | 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 detectionAntibodyEnzyme-linked immunosorbent assayIsopycnic centrifugationPorcine circovirus type 2Sucrose cushionFew 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.Departamento de Microbiologia e Imunologia Instituto de Biociências Universidade Estadual Paulista (Unesp)Instituto de Biotecnologia (Ibtec) UnespDepartamento de Medicina Veterinária Preventiva e Saúde Animal Faculdade de Medicina Veterinária e Zootecnia Universidade de São Paulo (USP), Av. Prof. Dr. Orlando Marques de Paiva 87Departamento de Microbiologia e Imunologia Instituto de Biociências Universidade Estadual Paulista (Unesp)Instituto de Biotecnologia (Ibtec) UnespUniversidade Estadual Paulista (Unesp)Universidade de São Paulo (USP)Cruz, Taís F. [UNESP]Kanashiro, Tatiana M. [UNESP]de Castro, Alessandra M.M.G.Baldin, Cintia M.Richtzenhain, Leonardo J.Araujo, João P. [UNESP]2018-12-11T17:10:29Z2018-12-11T17:10:29Z2016-01-01info:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/article1171-1177application/pdfhttp://dx.doi.org/10.1590/S0100-736X2016001200005Pesquisa Veterinaria Brasileira, v. 36, n. 12, p. 1171-1177, 2016.1678-51500100-736Xhttp://hdl.handle.net/11449/17431410.1590/S0100-736X2016001200005S0100-736X20160012011712-s2.0-85014774156S0100-736X2016001201171.pdfScopusreponame:Repositório Institucional da UNESPinstname:Universidade Estadual Paulista (UNESP)instacron:UNESPengPesquisa Veterinaria Brasileirainfo:eu-repo/semantics/openAccess2024-01-12T06:26:33Zoai:repositorio.unesp.br:11449/174314Repositório InstitucionalPUBhttp://repositorio.unesp.br/oai/requestopendoar:29462024-01-12T06:26:33Repositório Institucional da UNESP - Universidade Estadual Paulista (UNESP)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. [UNESP] Antibody Enzyme-linked immunosorbent assay Isopycnic centrifugation Porcine circovirus type 2 Sucrose cushion |
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. [UNESP] |
author_facet |
Cruz, Taís F. [UNESP] Kanashiro, Tatiana M. [UNESP] de Castro, Alessandra M.M.G. Baldin, Cintia M. Richtzenhain, Leonardo J. Araujo, João P. [UNESP] |
author_role |
author |
author2 |
Kanashiro, Tatiana M. [UNESP] de Castro, Alessandra M.M.G. Baldin, Cintia M. Richtzenhain, Leonardo J. Araujo, João P. [UNESP] |
author2_role |
author author author author author |
dc.contributor.none.fl_str_mv |
Universidade Estadual Paulista (Unesp) Universidade de São Paulo (USP) |
dc.contributor.author.fl_str_mv |
Cruz, Taís F. [UNESP] Kanashiro, Tatiana M. [UNESP] de Castro, Alessandra M.M.G. Baldin, Cintia M. Richtzenhain, Leonardo J. Araujo, João P. [UNESP] |
dc.subject.por.fl_str_mv |
Antibody Enzyme-linked immunosorbent assay Isopycnic centrifugation Porcine circovirus type 2 Sucrose cushion |
topic |
Antibody Enzyme-linked immunosorbent assay Isopycnic centrifugation Porcine circovirus type 2 Sucrose cushion |
description |
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 |
2016-01-01 2018-12-11T17:10:29Z 2018-12-11T17:10:29Z |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/article |
format |
article |
status_str |
publishedVersion |
dc.identifier.uri.fl_str_mv |
http://dx.doi.org/10.1590/S0100-736X2016001200005 Pesquisa Veterinaria Brasileira, v. 36, n. 12, p. 1171-1177, 2016. 1678-5150 0100-736X http://hdl.handle.net/11449/174314 10.1590/S0100-736X2016001200005 S0100-736X2016001201171 2-s2.0-85014774156 S0100-736X2016001201171.pdf |
url |
http://dx.doi.org/10.1590/S0100-736X2016001200005 http://hdl.handle.net/11449/174314 |
identifier_str_mv |
Pesquisa Veterinaria Brasileira, v. 36, n. 12, p. 1171-1177, 2016. 1678-5150 0100-736X 10.1590/S0100-736X2016001200005 S0100-736X2016001201171 2-s2.0-85014774156 S0100-736X2016001201171.pdf |
dc.language.iso.fl_str_mv |
eng |
language |
eng |
dc.relation.none.fl_str_mv |
Pesquisa Veterinaria Brasileira |
dc.rights.driver.fl_str_mv |
info:eu-repo/semantics/openAccess |
eu_rights_str_mv |
openAccess |
dc.format.none.fl_str_mv |
1171-1177 application/pdf |
dc.source.none.fl_str_mv |
Scopus reponame:Repositório Institucional da UNESP instname:Universidade Estadual Paulista (UNESP) instacron:UNESP |
instname_str |
Universidade Estadual Paulista (UNESP) |
instacron_str |
UNESP |
institution |
UNESP |
reponame_str |
Repositório Institucional da UNESP |
collection |
Repositório Institucional da UNESP |
repository.name.fl_str_mv |
Repositório Institucional da UNESP - Universidade Estadual Paulista (UNESP) |
repository.mail.fl_str_mv |
|
_version_ |
1799965596157214720 |