Desenvolvimento de um ELISA de bloqueio para o diagnóstico da neosporose bovina
Autor(a) principal: | |
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Data de Publicação: | 2014 |
Tipo de documento: | Dissertação |
Idioma: | por |
Título da fonte: | Repositório Institucional da UFPel - Guaiaca |
Texto Completo: | http://repositorio.ufpel.edu.br/handle/ri/2326 |
Resumo: | Neosporosis is a disease caused by the obligate intracellular parasite Neospora caninum, causing reproductive disorders, such abortions and sltillbirths in several animals, being considered a major cause of abortion in cattle worldwide. The serological standard diagnosis is indirect fluorescent-antibody test (IFAT), but due to morphological and antigenic similarities of N. caninum with other protozoa of the phylum Apicomplexa, such as Toxoplasma gondii and Sarcocystis spp., false positive results may occur. To improve the diagnosis of this disease and reduce cross-reactions with other coccidian, the use of specific recombinant proteins of N. caninum has been described. The Nc-p43 surface protein encoded by the gene NcSRS2 is specific of the parasite and is present in tachyzoites and bradyzoites of N. caninum. Indirect ELISA format assays have been developed for the diagnosis of neosporosis, however the blocking ELISA may present additional level of specificity. In this context, the objective of this study was develop and standardize a blocking ELISA (b-ELISA) for the diagnosis of bovine neosporosis. To this end, NcSRS2 gene, previously cloned into plasmid vector, was used to produce the recombinant protein (rNc-p43) in prokaryotic expression system Escherichia coli. The purified protein was used to produce a polyclonal antibody (pAb/rNc-p43) in rabbit and this was further purified and characterized by indirect ELISA. For the development of the b-ELISA were used a total of 152 cattle sera (71 negative and 81 positive) and 10 positive cattle sera for toxoplasmosis, all previously confirmed by IFI. As controls, a pool of all positive sera and a pool of all negative sera were used, in addition to a normal calf serum, confirmed by IFAT, used with negative reference sera. The percent inhibition for each sample was determined by comparing the optical density (OD) mean of test sera to the OD mean of negative reference sera. The cut-off value was determined as the percent inhibition of negative pool. The results suggest that the b-ELISA is a tool that can be used for diagnosis of bovine neosporosis, with a sensitivity of 98.7% and specificity of 88.7% when compared with the IFAT. Antibodies against T. gondii present in positive samples for toxoplasmosis were not able to recognize the rNc-p43. The b-ELISA showed that serum samples positive for N. caninum is able to prevent the binding of pAb/rNc-p43, enabling detect different class of antibodies in a single assay. This technique is |
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2014-08-20T14:31:30Z2014-04-092014-08-20T14:31:30Z2014-02-25SINNOTT, Francine Alves. Development of a blocking ELISA for the diagnosis of bovine neosporosis. 2014. 62 f. Dissertação (Mestrado em Biologia) - Universidade Federal de Pelotas, Pelotas, 2014.http://repositorio.ufpel.edu.br/handle/ri/2326Neosporosis is a disease caused by the obligate intracellular parasite Neospora caninum, causing reproductive disorders, such abortions and sltillbirths in several animals, being considered a major cause of abortion in cattle worldwide. The serological standard diagnosis is indirect fluorescent-antibody test (IFAT), but due to morphological and antigenic similarities of N. caninum with other protozoa of the phylum Apicomplexa, such as Toxoplasma gondii and Sarcocystis spp., false positive results may occur. To improve the diagnosis of this disease and reduce cross-reactions with other coccidian, the use of specific recombinant proteins of N. caninum has been described. The Nc-p43 surface protein encoded by the gene NcSRS2 is specific of the parasite and is present in tachyzoites and bradyzoites of N. caninum. Indirect ELISA format assays have been developed for the diagnosis of neosporosis, however the blocking ELISA may present additional level of specificity. In this context, the objective of this study was develop and standardize a blocking ELISA (b-ELISA) for the diagnosis of bovine neosporosis. To this end, NcSRS2 gene, previously cloned into plasmid vector, was used to produce the recombinant protein (rNc-p43) in prokaryotic expression system Escherichia coli. The purified protein was used to produce a polyclonal antibody (pAb/rNc-p43) in rabbit and this was further purified and characterized by indirect ELISA. For the development of the b-ELISA were used a total of 152 cattle sera (71 negative and 81 positive) and 10 positive cattle sera for toxoplasmosis, all previously confirmed by IFI. As controls, a pool of all positive sera and a pool of all negative sera were used, in addition to a normal calf serum, confirmed by IFAT, used with negative reference sera. The percent inhibition for each sample was determined by comparing the optical density (OD) mean of test sera to the OD mean of negative reference sera. The cut-off value was determined as the percent inhibition of negative pool. The results suggest that the b-ELISA is a tool that can be used for diagnosis of bovine neosporosis, with a sensitivity of 98.7% and specificity of 88.7% when compared with the IFAT. Antibodies against T. gondii present in positive samples for toxoplasmosis were not able to recognize the rNc-p43. The b-ELISA showed that serum samples positive for N. caninum is able to prevent the binding of pAb/rNc-p43, enabling detect different class of antibodies in a single assay. This technique isNeosporose é a doença causada pelo parasito intracelular obrigatório Neospora caninum, causando problemas reprodutivos, como abortos e mortalidade neonatal em diversos animais, sendo considerada uma das principais causas de abortos em bovinos em todo o mundo. O diagnóstico sorológico padrão é a imunofluorescência indireta (IFI), porém devido a semelhanças morfológicas e antigênicas de N. caninum com outros protozoários do filo Apicomplexa, como Toxoplasma gondii e Sarcocystis spp., pode ocorrer resultados falsos positivos. Para melhorar o diagnóstico desta parasitose e diminuir as reações cruzadas com outros coccídeos, à utilização de proteínas recombinantes específicas de N. caninum vem sendo descrita. A proteína de superfície Nc-p43 codificada pelo gene NcSRS2 é específica do parasito e está presente em taquizoítos e bradizoítos de N. caninum. Ensaios no formato ELISA indireto vêm sendo desenvolvidos para o diagnóstico da neosporose, no entanto os ELISAs de bloqueio podem apresentar nível adicional de especificidade. Neste contexto, o objetivo deste trabalho foi desenvolver e padronizar um ELISA no formato bloqueio (b-ELISA) para o diagnóstico da neosporose bovina. Para tal, o gene NcSRS2, previamente clonado em vetor plasmidial, foi utilizado para produzir a proteína recombinante (rNc-p43) em sistema de expressão procarioto Escherichia coli. A proteína purificada foi utilizada para produzir um anticorpo policlonal (pAb/rNc-p43) em coelho e este foi posteriormente purificado e caracterizado através de ELISA indireto. Para o desenvolvimento do b-ELISA foram utilizados um total de 152 soros bovinos (71 negativos e 81 positivos) e 10 soros bovinos positivos para toxoplasmose, todos previamente confirmados por IFI. Como controles, um pool de todos os soros positivos e um pool de todos os soros negativos foi utilizado, além de um soro normal de terneiro, confirmado através de IFI, utilizado como controle negativo referencial. O percentual de inibição de cada amostra foi determinado pela comparação da média da densidade óptica (DO) de cada soro teste com a média da DO do controle negativo referencial. O ponto de corte foi determinado pelo percentual de inibição do pool negativo. Os resultados obtidos sugerem que o b-ELISA é uma ferramenta que pode ser utilizada no diagnóstico da neosporose bovina, com sensibilidade de 98.7% e especificidade de 88.7%, quando comparado com a IFI. Os anticorpos contra T. gondii presentes nas amostras positivas para toxoplasmose não foram capazes de reconhecer a rNc-p43. O b-ELISA demonstrou que as amostras de soros positivas para N. caninum são capazes de impedir a ligação do pAb/rNc-p43, possibilitando a detecção de diferentes classes de anticorpos em um único ensaio. Esta técnica pode ser uma alternativa de triagem para a detecção de anticorpos contra N. caninum em populações de bovinos.application/pdfporUniversidade Federal de PelotasPrograma de Pós-Graduação em ParasitologiaUFPelBRBiologiaNeosporoseNeospora caninumELISADiagnósticoNc-p43NeosporosisNeospora caninumELISADiagnosticNc-p43CNPQ::CIENCIAS BIOLOGICAS::PARASITOLOGIADesenvolvimento de um ELISA de bloqueio para o diagnóstico da neosporose bovinaDevelopment of a blocking ELISA for the diagnosis of bovine neosporosisinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/masterThesishttp://lattes.cnpq.br/7091565633124789lattes.cnpq.br/8784620034013411Fernandes, Cláudia Pinho HartlebenSinnott, Francine Alvesinfo:eu-repo/semantics/openAccessreponame:Repositório Institucional da UFPel - Guaiacainstname:Universidade Federal de Pelotas (UFPEL)instacron:UFPELORIGINALdissertacao_francine_sinnott.pdfapplication/pdf966836http://guaiaca.ufpel.edu.br/xmlui/bitstream/123456789/2326/1/dissertacao_francine_sinnott.pdf31b75fbc3400564832438403aff0c6e0MD51open accessTEXTdissertacao_francine_sinnott.pdf.txtdissertacao_francine_sinnott.pdf.txtExtracted Texttext/plain112504http://guaiaca.ufpel.edu.br/xmlui/bitstream/123456789/2326/2/dissertacao_francine_sinnott.pdf.txt693503ca5efa76850a663f684ec61736MD52open accessTHUMBNAILdissertacao_francine_sinnott.pdf.jpgdissertacao_francine_sinnott.pdf.jpgGenerated Thumbnailimage/jpeg1221http://guaiaca.ufpel.edu.br/xmlui/bitstream/123456789/2326/3/dissertacao_francine_sinnott.pdf.jpg833af77688271c6d990eb7e5da7256f9MD53open access123456789/23262019-09-27 10:48:35.542open accessoai:guaiaca.ufpel.edu.br:123456789/2326Repositório InstitucionalPUBhttp://repositorio.ufpel.edu.br/oai/requestrippel@ufpel.edu.br || repositorio@ufpel.edu.br || aline.batista@ufpel.edu.bropendoar:2019-09-27T13:48:35Repositório Institucional da UFPel - Guaiaca - Universidade Federal de Pelotas (UFPEL)false |
dc.title.por.fl_str_mv |
Desenvolvimento de um ELISA de bloqueio para o diagnóstico da neosporose bovina |
dc.title.alternative.eng.fl_str_mv |
Development of a blocking ELISA for the diagnosis of bovine neosporosis |
title |
Desenvolvimento de um ELISA de bloqueio para o diagnóstico da neosporose bovina |
spellingShingle |
Desenvolvimento de um ELISA de bloqueio para o diagnóstico da neosporose bovina Sinnott, Francine Alves Neosporose Neospora caninum ELISA Diagnóstico Nc-p43 Neosporosis Neospora caninum ELISA Diagnostic Nc-p43 CNPQ::CIENCIAS BIOLOGICAS::PARASITOLOGIA |
title_short |
Desenvolvimento de um ELISA de bloqueio para o diagnóstico da neosporose bovina |
title_full |
Desenvolvimento de um ELISA de bloqueio para o diagnóstico da neosporose bovina |
title_fullStr |
Desenvolvimento de um ELISA de bloqueio para o diagnóstico da neosporose bovina |
title_full_unstemmed |
Desenvolvimento de um ELISA de bloqueio para o diagnóstico da neosporose bovina |
title_sort |
Desenvolvimento de um ELISA de bloqueio para o diagnóstico da neosporose bovina |
author |
Sinnott, Francine Alves |
author_facet |
Sinnott, Francine Alves |
author_role |
author |
dc.contributor.authorLattes.por.fl_str_mv |
http://lattes.cnpq.br/7091565633124789 |
dc.contributor.advisorLattes.por.fl_str_mv |
lattes.cnpq.br/8784620034013411 |
dc.contributor.advisor1.fl_str_mv |
Fernandes, Cláudia Pinho Hartleben |
dc.contributor.author.fl_str_mv |
Sinnott, Francine Alves |
contributor_str_mv |
Fernandes, Cláudia Pinho Hartleben |
dc.subject.por.fl_str_mv |
Neosporose Neospora caninum ELISA Diagnóstico Nc-p43 |
topic |
Neosporose Neospora caninum ELISA Diagnóstico Nc-p43 Neosporosis Neospora caninum ELISA Diagnostic Nc-p43 CNPQ::CIENCIAS BIOLOGICAS::PARASITOLOGIA |
dc.subject.eng.fl_str_mv |
Neosporosis Neospora caninum ELISA Diagnostic Nc-p43 |
dc.subject.cnpq.fl_str_mv |
CNPQ::CIENCIAS BIOLOGICAS::PARASITOLOGIA |
description |
Neosporosis is a disease caused by the obligate intracellular parasite Neospora caninum, causing reproductive disorders, such abortions and sltillbirths in several animals, being considered a major cause of abortion in cattle worldwide. The serological standard diagnosis is indirect fluorescent-antibody test (IFAT), but due to morphological and antigenic similarities of N. caninum with other protozoa of the phylum Apicomplexa, such as Toxoplasma gondii and Sarcocystis spp., false positive results may occur. To improve the diagnosis of this disease and reduce cross-reactions with other coccidian, the use of specific recombinant proteins of N. caninum has been described. The Nc-p43 surface protein encoded by the gene NcSRS2 is specific of the parasite and is present in tachyzoites and bradyzoites of N. caninum. Indirect ELISA format assays have been developed for the diagnosis of neosporosis, however the blocking ELISA may present additional level of specificity. In this context, the objective of this study was develop and standardize a blocking ELISA (b-ELISA) for the diagnosis of bovine neosporosis. To this end, NcSRS2 gene, previously cloned into plasmid vector, was used to produce the recombinant protein (rNc-p43) in prokaryotic expression system Escherichia coli. The purified protein was used to produce a polyclonal antibody (pAb/rNc-p43) in rabbit and this was further purified and characterized by indirect ELISA. For the development of the b-ELISA were used a total of 152 cattle sera (71 negative and 81 positive) and 10 positive cattle sera for toxoplasmosis, all previously confirmed by IFI. As controls, a pool of all positive sera and a pool of all negative sera were used, in addition to a normal calf serum, confirmed by IFAT, used with negative reference sera. The percent inhibition for each sample was determined by comparing the optical density (OD) mean of test sera to the OD mean of negative reference sera. The cut-off value was determined as the percent inhibition of negative pool. The results suggest that the b-ELISA is a tool that can be used for diagnosis of bovine neosporosis, with a sensitivity of 98.7% and specificity of 88.7% when compared with the IFAT. Antibodies against T. gondii present in positive samples for toxoplasmosis were not able to recognize the rNc-p43. The b-ELISA showed that serum samples positive for N. caninum is able to prevent the binding of pAb/rNc-p43, enabling detect different class of antibodies in a single assay. This technique is |
publishDate |
2014 |
dc.date.accessioned.fl_str_mv |
2014-08-20T14:31:30Z |
dc.date.available.fl_str_mv |
2014-04-09 2014-08-20T14:31:30Z |
dc.date.issued.fl_str_mv |
2014-02-25 |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/masterThesis |
format |
masterThesis |
status_str |
publishedVersion |
dc.identifier.citation.fl_str_mv |
SINNOTT, Francine Alves. Development of a blocking ELISA for the diagnosis of bovine neosporosis. 2014. 62 f. Dissertação (Mestrado em Biologia) - Universidade Federal de Pelotas, Pelotas, 2014. |
dc.identifier.uri.fl_str_mv |
http://repositorio.ufpel.edu.br/handle/ri/2326 |
identifier_str_mv |
SINNOTT, Francine Alves. Development of a blocking ELISA for the diagnosis of bovine neosporosis. 2014. 62 f. Dissertação (Mestrado em Biologia) - Universidade Federal de Pelotas, Pelotas, 2014. |
url |
http://repositorio.ufpel.edu.br/handle/ri/2326 |
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