Padronização da quantificação do fator de crescimento semelhante à insulina I (IGF-I) em plasma bovino por ELISA1
Autor(a) principal: | |
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Data de Publicação: | 2017 |
Outros Autores: | |
Tipo de documento: | Artigo |
Idioma: | por |
Título da fonte: | Repositório Institucional da UNESP |
Texto Completo: | http://dx.doi.org/10.1590/S0100-736X2017001200030 http://hdl.handle.net/11449/179688 |
Resumo: | This study aimed to standardize an in house competitive enzyme-linked immunosorbent assay (cELISA) to determine plasma concentrations of total insulin-like growth factor I (IGF-I) for the bovine specie using the amplification biotin-streptavidin peroxidase system. The IGF-I was extracted from insulin-like growth factor binding proteins (IGFBPs) using the acidified glycine buffer followed by the pH neutralization with sodium hydroxide. The microplates were coated with anti-rabbit IgG, thereafter the measurements were carried out using two approaches, one with a prior incubation of samples with the anti-h-IGF-I antibody and another without previous incubation (simultaneous addition of IGF-I and biotinylated sample). The best results were obtained using the method without the prior incubation, using the following combination of reagents: microplates were coated with 0.25μg/well of anti- rabbit IgG, the specific antibody at a dilution of 1:250,000 and 0.06ng/well of biotinylated IGF-I. The in house methodology showed sensitivity of 50ng/ml, a correlation between doses of 0.945 when compared to a commercial method. In addition, after 33 assays (quantification of 1114 samples) the proposed methodology presented a good precision, with inter-assay variation coefficients of 12.94% and 20.71% for the high and low controls, respectively. Finally, we concluded that ELISA method for the quantification of total IGF-I using the system biotin-streptavidin-peroxidase amplification in a competitive assay is established and is presented as a useful tool for studies aimed at monitoring the IGF-I concentrations. |
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Padronização da quantificação do fator de crescimento semelhante à insulina I (IGF-I) em plasma bovino por ELISA1Standardization of ELISA for the measurement of Insulin-Like Growth Factor I (IGF-I) in bovine plasmBovineELISAHormoneIGF-IInsulin-like growth factor IPlasmValidationThis study aimed to standardize an in house competitive enzyme-linked immunosorbent assay (cELISA) to determine plasma concentrations of total insulin-like growth factor I (IGF-I) for the bovine specie using the amplification biotin-streptavidin peroxidase system. The IGF-I was extracted from insulin-like growth factor binding proteins (IGFBPs) using the acidified glycine buffer followed by the pH neutralization with sodium hydroxide. The microplates were coated with anti-rabbit IgG, thereafter the measurements were carried out using two approaches, one with a prior incubation of samples with the anti-h-IGF-I antibody and another without previous incubation (simultaneous addition of IGF-I and biotinylated sample). The best results were obtained using the method without the prior incubation, using the following combination of reagents: microplates were coated with 0.25μg/well of anti- rabbit IgG, the specific antibody at a dilution of 1:250,000 and 0.06ng/well of biotinylated IGF-I. The in house methodology showed sensitivity of 50ng/ml, a correlation between doses of 0.945 when compared to a commercial method. In addition, after 33 assays (quantification of 1114 samples) the proposed methodology presented a good precision, with inter-assay variation coefficients of 12.94% and 20.71% for the high and low controls, respectively. Finally, we concluded that ELISA method for the quantification of total IGF-I using the system biotin-streptavidin-peroxidase amplification in a competitive assay is established and is presented as a useful tool for studies aimed at monitoring the IGF-I concentrations.Departamento de Apoio a Produção e Saúde Animal Faculdade de Medicina Veterinária Universidade Estadual Paulista Júlio de Mesquita Filho (Unesp), Rua Clóvis Pestana 793, Dona AméliaDepartamento de Apoio a Produção e Saúde Animal Faculdade de Medicina Veterinária Universidade Estadual Paulista Júlio de Mesquita Filho (Unesp), Rua Clóvis Pestana 793, Dona AméliaUniversidade Estadual Paulista (Unesp)Maioli, Marcos Antonio [UNESP]Nogueira, Guilherme de Paula [UNESP]2018-12-11T17:36:22Z2018-12-11T17:36:22Z2017-12-01info:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/article1545-1553application/pdfhttp://dx.doi.org/10.1590/S0100-736X2017001200030Pesquisa Veterinaria Brasileira, v. 37, n. 12, p. 1545-1553, 2017.1678-51500100-736Xhttp://hdl.handle.net/11449/17968810.1590/S0100-736X2017001200030S0100-736X20170012015452-s2.0-85044202650S0100-736X2017001201545.pdfScopusreponame:Repositório Institucional da UNESPinstname:Universidade Estadual Paulista (UNESP)instacron:UNESPporPesquisa Veterinaria Brasileirainfo:eu-repo/semantics/openAccess2024-09-04T19:15:39Zoai:repositorio.unesp.br:11449/179688Repositório InstitucionalPUBhttp://repositorio.unesp.br/oai/requestrepositoriounesp@unesp.bropendoar:29462024-09-04T19:15:39Repositório Institucional da UNESP - Universidade Estadual Paulista (UNESP)false |
dc.title.none.fl_str_mv |
Padronização da quantificação do fator de crescimento semelhante à insulina I (IGF-I) em plasma bovino por ELISA1 Standardization of ELISA for the measurement of Insulin-Like Growth Factor I (IGF-I) in bovine plasm |
title |
Padronização da quantificação do fator de crescimento semelhante à insulina I (IGF-I) em plasma bovino por ELISA1 |
spellingShingle |
Padronização da quantificação do fator de crescimento semelhante à insulina I (IGF-I) em plasma bovino por ELISA1 Maioli, Marcos Antonio [UNESP] Bovine ELISA Hormone IGF-I Insulin-like growth factor I Plasm Validation |
title_short |
Padronização da quantificação do fator de crescimento semelhante à insulina I (IGF-I) em plasma bovino por ELISA1 |
title_full |
Padronização da quantificação do fator de crescimento semelhante à insulina I (IGF-I) em plasma bovino por ELISA1 |
title_fullStr |
Padronização da quantificação do fator de crescimento semelhante à insulina I (IGF-I) em plasma bovino por ELISA1 |
title_full_unstemmed |
Padronização da quantificação do fator de crescimento semelhante à insulina I (IGF-I) em plasma bovino por ELISA1 |
title_sort |
Padronização da quantificação do fator de crescimento semelhante à insulina I (IGF-I) em plasma bovino por ELISA1 |
author |
Maioli, Marcos Antonio [UNESP] |
author_facet |
Maioli, Marcos Antonio [UNESP] Nogueira, Guilherme de Paula [UNESP] |
author_role |
author |
author2 |
Nogueira, Guilherme de Paula [UNESP] |
author2_role |
author |
dc.contributor.none.fl_str_mv |
Universidade Estadual Paulista (Unesp) |
dc.contributor.author.fl_str_mv |
Maioli, Marcos Antonio [UNESP] Nogueira, Guilherme de Paula [UNESP] |
dc.subject.por.fl_str_mv |
Bovine ELISA Hormone IGF-I Insulin-like growth factor I Plasm Validation |
topic |
Bovine ELISA Hormone IGF-I Insulin-like growth factor I Plasm Validation |
description |
This study aimed to standardize an in house competitive enzyme-linked immunosorbent assay (cELISA) to determine plasma concentrations of total insulin-like growth factor I (IGF-I) for the bovine specie using the amplification biotin-streptavidin peroxidase system. The IGF-I was extracted from insulin-like growth factor binding proteins (IGFBPs) using the acidified glycine buffer followed by the pH neutralization with sodium hydroxide. The microplates were coated with anti-rabbit IgG, thereafter the measurements were carried out using two approaches, one with a prior incubation of samples with the anti-h-IGF-I antibody and another without previous incubation (simultaneous addition of IGF-I and biotinylated sample). The best results were obtained using the method without the prior incubation, using the following combination of reagents: microplates were coated with 0.25μg/well of anti- rabbit IgG, the specific antibody at a dilution of 1:250,000 and 0.06ng/well of biotinylated IGF-I. The in house methodology showed sensitivity of 50ng/ml, a correlation between doses of 0.945 when compared to a commercial method. In addition, after 33 assays (quantification of 1114 samples) the proposed methodology presented a good precision, with inter-assay variation coefficients of 12.94% and 20.71% for the high and low controls, respectively. Finally, we concluded that ELISA method for the quantification of total IGF-I using the system biotin-streptavidin-peroxidase amplification in a competitive assay is established and is presented as a useful tool for studies aimed at monitoring the IGF-I concentrations. |
publishDate |
2017 |
dc.date.none.fl_str_mv |
2017-12-01 2018-12-11T17:36:22Z 2018-12-11T17:36:22Z |
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/S0100-736X2017001200030 Pesquisa Veterinaria Brasileira, v. 37, n. 12, p. 1545-1553, 2017. 1678-5150 0100-736X http://hdl.handle.net/11449/179688 10.1590/S0100-736X2017001200030 S0100-736X2017001201545 2-s2.0-85044202650 S0100-736X2017001201545.pdf |
url |
http://dx.doi.org/10.1590/S0100-736X2017001200030 http://hdl.handle.net/11449/179688 |
identifier_str_mv |
Pesquisa Veterinaria Brasileira, v. 37, n. 12, p. 1545-1553, 2017. 1678-5150 0100-736X 10.1590/S0100-736X2017001200030 S0100-736X2017001201545 2-s2.0-85044202650 S0100-736X2017001201545.pdf |
dc.language.iso.fl_str_mv |
por |
language |
por |
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 |
1545-1553 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 |
repositoriounesp@unesp.br |
_version_ |
1810021396294664192 |