Desenvolvimento de ensaio imunoquímico para detecção de doping com eritropoetina
Autor(a) principal: | |
---|---|
Data de Publicação: | 2013 |
Tipo de documento: | Tese |
Idioma: | por |
Título da fonte: | Repositório Institucional da UFPel - Guaiaca |
Texto Completo: | http://guaiaca.ufpel.edu.br/handle/123456789/1225 |
Resumo: | The history of sports competition has been related to the use of methods for physical training associated with physiological methods to increase athlete performance. Ethical issues involving the high performance sport and doping are confused with the history of competitive sports. The World Anti - Doping Agency (WADA) prohibits the use of substances or methods to artificially increase sports performance. Currently, 258 substances are listed by WADA and athletes around the world are subjected to tests proving the non-use of doping. EPO is a glycoprotein hormone that has as its main physiological effect induction of erythropoiesis and thereby improving the capacity to transport oxygen in the blood. For these reason EPO has been included in WADA list. The analytical differentiation of endogenous erythropoietin from its recombinant counterpart, using isoelectric focusing and double blotting is a milestone in the detection of doping with recombinant erythropoietin. However, several analogs of the original recombinant EPO are not easily detectable by standard IEF method, requiring the development of alternatives for the detection of doping. In order to improve the current methods of EPO detection, monoclonal and polyclonal antibodies against EPO were obtained and used in various techniques for detection of EPO in biological samples. However, the specificity of these antibodies has been quite controversial and discussed. Therefore, it is necessary to obtain antibodies capable of reacting specifically with the EPO. In this study, rHuEPO was inoculated in New Zealand rabbits to generate a polyclonal antibody (pAb anti-rHuEPO). The pAb was characterized for its potential in detecting rHuEPO using different approaches. The pAb anti- rHuEPO identified the expression of recombinant protein in eukaryotic cells and was able to detect rHuEPO in a suspension at 0.1 μg/mL, showing its potential as a tool for detection of doping by rHuEPO. |
id |
UFPL_cedd2f37b0ce64518f1fe2f5c1016c18 |
---|---|
oai_identifier_str |
oai:guaiaca.ufpel.edu.br:123456789/1225 |
network_acronym_str |
UFPL |
network_name_str |
Repositório Institucional da UFPel - Guaiaca |
repository_id_str |
|
spelling |
2014-08-20T13:32:48Z2014-01-272014-08-20T13:32:48Z2013-10-17COLLARES, Thais Farias. Development of immunochemical assay for detection of doping with erythropoietin. 2013. 59 f. Tese (Doutorado em Biotecnologia) - Universidade Federal de Pelotas, Pelotas, 2013.http://guaiaca.ufpel.edu.br/handle/123456789/1225The history of sports competition has been related to the use of methods for physical training associated with physiological methods to increase athlete performance. Ethical issues involving the high performance sport and doping are confused with the history of competitive sports. The World Anti - Doping Agency (WADA) prohibits the use of substances or methods to artificially increase sports performance. Currently, 258 substances are listed by WADA and athletes around the world are subjected to tests proving the non-use of doping. EPO is a glycoprotein hormone that has as its main physiological effect induction of erythropoiesis and thereby improving the capacity to transport oxygen in the blood. For these reason EPO has been included in WADA list. The analytical differentiation of endogenous erythropoietin from its recombinant counterpart, using isoelectric focusing and double blotting is a milestone in the detection of doping with recombinant erythropoietin. However, several analogs of the original recombinant EPO are not easily detectable by standard IEF method, requiring the development of alternatives for the detection of doping. In order to improve the current methods of EPO detection, monoclonal and polyclonal antibodies against EPO were obtained and used in various techniques for detection of EPO in biological samples. However, the specificity of these antibodies has been quite controversial and discussed. Therefore, it is necessary to obtain antibodies capable of reacting specifically with the EPO. In this study, rHuEPO was inoculated in New Zealand rabbits to generate a polyclonal antibody (pAb anti-rHuEPO). The pAb was characterized for its potential in detecting rHuEPO using different approaches. The pAb anti- rHuEPO identified the expression of recombinant protein in eukaryotic cells and was able to detect rHuEPO in a suspension at 0.1 μg/mL, showing its potential as a tool for detection of doping by rHuEPO.A história da competição esportiva sempre esteve relacionada à utilização de metodologias de treinamento físico associadas a métodos de incremento fisiológico do atleta, visando sua máxima performance. Questões éticas envolvendo o esporte de alto rendimento e o doping se confundem com a própria história do esporte competitivo. A Agência Mundial Anti-Doping (WADA) proíbe o uso de substâncias ou métodos capazes de aumentar artificialmente o desempenho esportivo. Atualmente, 258 substâncias estão na lista da WADA e atletas do mundo inteiro são submetidos a testes comprobatórios da não utilização do doping. Entre essas substâncias ilícitas para uso por atletas destaca-se a eritropoetina (EPO). A EPO é um hormônio glicoproteico que possui como principal efeito fisiológico a indução da eritropoiese e consequente melhoria da capacidade de transporte de oxigênio no sangue. A diferenciação analítica da eritropoetina endógena produzida a partir de sua contraparte recombinante usando focalização isoelétrica e duplo blotting é um marco na detecção do doping com eritropoetina recombinante. Anticorpos monoclonais e policlonais específicos anti-EPO foram obtidos e utilizados em várias técnicas para a detecção da EPO em amostras biológicas a fim de melhorar os métodos atuais de detecção. Contudo, a especificidade destes anticorpos tem sido bastante controversa e discutida. Portanto, a obtenção de anticorpos específicos capazes de reagir especificamente com a EPO faz-se necessária. Neste estudo, a rHuEPO foi utilizada para imunizar coelhos New Zealand para gerar um anticorpo policlonal (pAb anti-rHuEPO). O pAb foi caracterizado quanto ao seu potencial na detecção da rHuEPO usando diferentes metodologias. O pAb anti-rHuEPO identificou a expressão da proteína recombinante em células eucarióticas e foi capaz de detectar rHuEPO em suspensão até 0,1 μg/mL, comprovando seu potencial como ferramenta para detecção do doping por rHuEPO.application/pdfporUniversidade Federal de PelotasPrograma de Pós-Graduação em BiotecnologiaUFPelBRBiotecnologiarHuEPOErythropoietinDoping controlAntibodiesImmunoassayspAbrHuEPOEritropoetinaAnti-dopingAnticorposImunoensaiospAbCNPQ::CIENCIAS BIOLOGICAS::IMUNOLOGIADesenvolvimento de ensaio imunoquímico para detecção de doping com eritropoetinaDevelopment of immunochemical assay for detection of doping with erythropoietininfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/doctoralThesishttp://lattes.cnpq.br/4251608907325877lattes.cnpq.br/8784620034013411Fernandes, Cláudia Pinho HartlebenCollares, Thais Fariasinfo:eu-repo/semantics/openAccessreponame:Repositório Institucional da UFPel - Guaiacainstname:Universidade Federal de Pelotas (UFPEL)instacron:UFPELORIGINALtese_thais_farias_collares.pdfapplication/pdf1094762http://guaiaca.ufpel.edu.br/xmlui/bitstream/123456789/1225/1/tese_thais_farias_collares.pdf647dd933472bf9127c0164ee875617eaMD51open accessTEXTtese_thais_farias_collares.pdf.txttese_thais_farias_collares.pdf.txtExtracted Texttext/plain80592http://guaiaca.ufpel.edu.br/xmlui/bitstream/123456789/1225/2/tese_thais_farias_collares.pdf.txt2d99f94180e3fae564fe1583015de906MD52open accessTHUMBNAILtese_thais_farias_collares.pdf.jpgtese_thais_farias_collares.pdf.jpgGenerated Thumbnailimage/jpeg1748http://guaiaca.ufpel.edu.br/xmlui/bitstream/123456789/1225/3/tese_thais_farias_collares.pdf.jpgec2e28e1d88bf526dba6bfce2ab3b643MD53open access123456789/12252019-08-23 10:10:20.065open accessoai:guaiaca.ufpel.edu.br:123456789/1225Repositório InstitucionalPUBhttp://repositorio.ufpel.edu.br/oai/requestrippel@ufpel.edu.br || repositorio@ufpel.edu.br || aline.batista@ufpel.edu.bropendoar:2019-08-23T13:10:20Repositório Institucional da UFPel - Guaiaca - Universidade Federal de Pelotas (UFPEL)false |
dc.title.por.fl_str_mv |
Desenvolvimento de ensaio imunoquímico para detecção de doping com eritropoetina |
dc.title.alternative.eng.fl_str_mv |
Development of immunochemical assay for detection of doping with erythropoietin |
title |
Desenvolvimento de ensaio imunoquímico para detecção de doping com eritropoetina |
spellingShingle |
Desenvolvimento de ensaio imunoquímico para detecção de doping com eritropoetina Collares, Thais Farias rHuEPO Erythropoietin Doping control Antibodies Immunoassays pAb rHuEPO Eritropoetina Anti-doping Anticorpos Imunoensaios pAb CNPQ::CIENCIAS BIOLOGICAS::IMUNOLOGIA |
title_short |
Desenvolvimento de ensaio imunoquímico para detecção de doping com eritropoetina |
title_full |
Desenvolvimento de ensaio imunoquímico para detecção de doping com eritropoetina |
title_fullStr |
Desenvolvimento de ensaio imunoquímico para detecção de doping com eritropoetina |
title_full_unstemmed |
Desenvolvimento de ensaio imunoquímico para detecção de doping com eritropoetina |
title_sort |
Desenvolvimento de ensaio imunoquímico para detecção de doping com eritropoetina |
author |
Collares, Thais Farias |
author_facet |
Collares, Thais Farias |
author_role |
author |
dc.contributor.authorLattes.por.fl_str_mv |
http://lattes.cnpq.br/4251608907325877 |
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 |
Collares, Thais Farias |
contributor_str_mv |
Fernandes, Cláudia Pinho Hartleben |
dc.subject.eng.fl_str_mv |
rHuEPO Erythropoietin Doping control Antibodies Immunoassays pAb |
topic |
rHuEPO Erythropoietin Doping control Antibodies Immunoassays pAb rHuEPO Eritropoetina Anti-doping Anticorpos Imunoensaios pAb CNPQ::CIENCIAS BIOLOGICAS::IMUNOLOGIA |
dc.subject.por.fl_str_mv |
rHuEPO Eritropoetina Anti-doping Anticorpos Imunoensaios pAb |
dc.subject.cnpq.fl_str_mv |
CNPQ::CIENCIAS BIOLOGICAS::IMUNOLOGIA |
description |
The history of sports competition has been related to the use of methods for physical training associated with physiological methods to increase athlete performance. Ethical issues involving the high performance sport and doping are confused with the history of competitive sports. The World Anti - Doping Agency (WADA) prohibits the use of substances or methods to artificially increase sports performance. Currently, 258 substances are listed by WADA and athletes around the world are subjected to tests proving the non-use of doping. EPO is a glycoprotein hormone that has as its main physiological effect induction of erythropoiesis and thereby improving the capacity to transport oxygen in the blood. For these reason EPO has been included in WADA list. The analytical differentiation of endogenous erythropoietin from its recombinant counterpart, using isoelectric focusing and double blotting is a milestone in the detection of doping with recombinant erythropoietin. However, several analogs of the original recombinant EPO are not easily detectable by standard IEF method, requiring the development of alternatives for the detection of doping. In order to improve the current methods of EPO detection, monoclonal and polyclonal antibodies against EPO were obtained and used in various techniques for detection of EPO in biological samples. However, the specificity of these antibodies has been quite controversial and discussed. Therefore, it is necessary to obtain antibodies capable of reacting specifically with the EPO. In this study, rHuEPO was inoculated in New Zealand rabbits to generate a polyclonal antibody (pAb anti-rHuEPO). The pAb was characterized for its potential in detecting rHuEPO using different approaches. The pAb anti- rHuEPO identified the expression of recombinant protein in eukaryotic cells and was able to detect rHuEPO in a suspension at 0.1 μg/mL, showing its potential as a tool for detection of doping by rHuEPO. |
publishDate |
2013 |
dc.date.issued.fl_str_mv |
2013-10-17 |
dc.date.accessioned.fl_str_mv |
2014-08-20T13:32:48Z |
dc.date.available.fl_str_mv |
2014-01-27 2014-08-20T13:32:48Z |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/doctoralThesis |
format |
doctoralThesis |
status_str |
publishedVersion |
dc.identifier.citation.fl_str_mv |
COLLARES, Thais Farias. Development of immunochemical assay for detection of doping with erythropoietin. 2013. 59 f. Tese (Doutorado em Biotecnologia) - Universidade Federal de Pelotas, Pelotas, 2013. |
dc.identifier.uri.fl_str_mv |
http://guaiaca.ufpel.edu.br/handle/123456789/1225 |
identifier_str_mv |
COLLARES, Thais Farias. Development of immunochemical assay for detection of doping with erythropoietin. 2013. 59 f. Tese (Doutorado em Biotecnologia) - Universidade Federal de Pelotas, Pelotas, 2013. |
url |
http://guaiaca.ufpel.edu.br/handle/123456789/1225 |
dc.language.iso.fl_str_mv |
por |
language |
por |
dc.rights.driver.fl_str_mv |
info:eu-repo/semantics/openAccess |
eu_rights_str_mv |
openAccess |
dc.format.none.fl_str_mv |
application/pdf |
dc.publisher.none.fl_str_mv |
Universidade Federal de Pelotas |
dc.publisher.program.fl_str_mv |
Programa de Pós-Graduação em Biotecnologia |
dc.publisher.initials.fl_str_mv |
UFPel |
dc.publisher.country.fl_str_mv |
BR |
dc.publisher.department.fl_str_mv |
Biotecnologia |
publisher.none.fl_str_mv |
Universidade Federal de Pelotas |
dc.source.none.fl_str_mv |
reponame:Repositório Institucional da UFPel - Guaiaca instname:Universidade Federal de Pelotas (UFPEL) instacron:UFPEL |
instname_str |
Universidade Federal de Pelotas (UFPEL) |
instacron_str |
UFPEL |
institution |
UFPEL |
reponame_str |
Repositório Institucional da UFPel - Guaiaca |
collection |
Repositório Institucional da UFPel - Guaiaca |
bitstream.url.fl_str_mv |
http://guaiaca.ufpel.edu.br/xmlui/bitstream/123456789/1225/1/tese_thais_farias_collares.pdf http://guaiaca.ufpel.edu.br/xmlui/bitstream/123456789/1225/2/tese_thais_farias_collares.pdf.txt http://guaiaca.ufpel.edu.br/xmlui/bitstream/123456789/1225/3/tese_thais_farias_collares.pdf.jpg |
bitstream.checksum.fl_str_mv |
647dd933472bf9127c0164ee875617ea 2d99f94180e3fae564fe1583015de906 ec2e28e1d88bf526dba6bfce2ab3b643 |
bitstream.checksumAlgorithm.fl_str_mv |
MD5 MD5 MD5 |
repository.name.fl_str_mv |
Repositório Institucional da UFPel - Guaiaca - Universidade Federal de Pelotas (UFPEL) |
repository.mail.fl_str_mv |
rippel@ufpel.edu.br || repositorio@ufpel.edu.br || aline.batista@ufpel.edu.br |
_version_ |
1801846936238555136 |