Development of a fluorescent based immunosensor for the serodiagnosis of canine Leishmaniasis combining immunomagnetic separation and flow cytometry.
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Publication Date: | 2013 |
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Format: | Article |
Language: | eng |
Source: | Repositório Institucional da UFOP |
Download full: | http://www.repositorio.ufop.br/handle/123456789/4264 https://doi.org/10.1371/journal.pntd.0002371 |
Summary: | An accurate diagnosis is essential for the control of infectious diseases. In the search for effective and efficient tests, biosensors have increasingly been exploited for the development of new and highly sensitive diagnostic methods. Here, we describe a new fluorescent based immunosensor comprising magnetic polymer microspheres coated with recombinant antigens to improve the detection of specific antibodies generated during an infectious disease. As a challenging model, we used canine leishmaniasis due to the unsatisfactory sensitivity associated with the detection of infection in asymptomatic animals where the levels of pathogen-specific antibodies are scarce. Methodology: Ni-NTA magnetic microspheres with 1,7 mm and 8,07 mm were coated with the Leishmania recombinant proteins LicTXNPx and rK39, respectively. A mixture of equal proportions of both recombinant protein-coated microspheres was used to recognize and specifically bind anti-rK39 and anti-LicTNXPx antibodies present in serum samples of infected dogs. The microspheres were recovered by magnetic separation and the percentage of fluorescent positive microspheres was quantified by flow cytometry. Principal Findings: A clinical evaluation carried out with 129 dog serum samples using the antigen combination demonstrated a sensitivity of 98,8% with a specificity of 94,4%. rK39 antigen alone demonstrated a higher sensitivity for symptomatic dogs (96,9%), while LicTXNPx antigen showed a higher sensitivity for asymptomatic (94,4%). Conclusions: Overall, our results demonstrated the potential of a magnetic microsphere associated flow cytometry methodology as a viable tool for highly sensitive laboratorial serodiagnosis of both clinical and subclinical forms of canine leishmaniasis. |
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Sousa, SusanaCardoso, LuísReed, Steven G.Reis, Alexandre BarbosaMartins Filho, Olindo AssisSilvestre, RicardoSilva, Anabela Cordeiro da2015-01-19T16:42:44Z2015-01-19T16:42:44Z2013SOUSA, S. et al. Development of a fluorescent based immunosensor for the serodiagnosis of canine Leishmaniasis combining immunomagnetic separation and flow cytometry. PLoS Neglected Tropical Diseases, v. 7, p. e2371, 2013. Disponível em: <http://www.plosntds.org/article/info%3Adoi%2F10.1371%2Fjournal.pntd.0002371>. Aceso em: 12 ago. 2014.1935-2735http://www.repositorio.ufop.br/handle/123456789/4264https://doi.org/10.1371/journal.pntd.0002371An accurate diagnosis is essential for the control of infectious diseases. In the search for effective and efficient tests, biosensors have increasingly been exploited for the development of new and highly sensitive diagnostic methods. Here, we describe a new fluorescent based immunosensor comprising magnetic polymer microspheres coated with recombinant antigens to improve the detection of specific antibodies generated during an infectious disease. As a challenging model, we used canine leishmaniasis due to the unsatisfactory sensitivity associated with the detection of infection in asymptomatic animals where the levels of pathogen-specific antibodies are scarce. Methodology: Ni-NTA magnetic microspheres with 1,7 mm and 8,07 mm were coated with the Leishmania recombinant proteins LicTXNPx and rK39, respectively. A mixture of equal proportions of both recombinant protein-coated microspheres was used to recognize and specifically bind anti-rK39 and anti-LicTNXPx antibodies present in serum samples of infected dogs. The microspheres were recovered by magnetic separation and the percentage of fluorescent positive microspheres was quantified by flow cytometry. Principal Findings: A clinical evaluation carried out with 129 dog serum samples using the antigen combination demonstrated a sensitivity of 98,8% with a specificity of 94,4%. rK39 antigen alone demonstrated a higher sensitivity for symptomatic dogs (96,9%), while LicTXNPx antigen showed a higher sensitivity for asymptomatic (94,4%). Conclusions: Overall, our results demonstrated the potential of a magnetic microsphere associated flow cytometry methodology as a viable tool for highly sensitive laboratorial serodiagnosis of both clinical and subclinical forms of canine leishmaniasis.Flow cytometryCanine leishmaniasisDevelopment of a fluorescent based immunosensor for the serodiagnosis of canine Leishmaniasis combining immunomagnetic separation and flow cytometry.info:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/articleOs trabalhos publicados na Plos one estão sob Licença Creative Commons que permite copiar, distribuir e transmitir o trabalho, desde que sejam citados o autor e licenciante. Não permite o uso para fins comerciais nem a adaptação. Fonte: Plos one <https://www.plos.org/open-access>. Acesso em: 03 jan. 2017.info:eu-repo/semantics/openAccessengreponame:Repositório Institucional da UFOPinstname:Universidade Federal de Ouro Preto (UFOP)instacron:UFOPLICENSElicense.txtlicense.txttext/plain; charset=utf-82636http://www.repositorio.ufop.br/bitstream/123456789/4264/2/license.txtc2ffdd99e58acf69202dff00d361f23aMD52ORIGINALARTIGO_DevelopmentFluorescent Based.pdfARTIGO_DevelopmentFluorescent Based.pdfapplication/pdf469540http://www.repositorio.ufop.br/bitstream/123456789/4264/1/ARTIGO_DevelopmentFluorescent%20Based.pdf4dc4921bb36c645b7a5410b15247e8d8MD51123456789/42642019-06-10 14:34:58.833oai:localhost:123456789/4264PGh0bWw+Cjxib2R5Pgo8ZGl2IGFsaWduPSJqdXN0aWZ5Ij48c3Ryb25nPkxpY2VuJmNjZWRpbDthIGRvIFJlcG9zaXQmb2FjdXRlO3JpbyBJbnN0aXR1Y2lvbmFsIGRhIFVuaXZlcnNpZGFkZSBGZWRlcmFsIGRlIE91cm8gUHJldG88L3N0cm9uZz4KICA8YnI+CiAgPGJyPgogIEFvIGNvbmNvcmRhciBjb20gZXN0YSBsaWNlbiZjY2VkaWw7YSwgdm9jJmVjaXJjOyhzKSBhdXRvcihlcykgb3UgdGl0dWxhcihlcykgZG9zIGRpcmVpdG9zIGF1dG9yYWlzIGRhIG9icmEgYXF1aSBkZXNjcml0YSBjb25jZWRlKG0pICZhZ3JhdmU7CiAgPGJyPgogIFVuaXZlcnNpZGFkZSBGZWRlcmFsIGRlIE91cm8gUHJldG8gKFVGT1ApIGdlc3RvcmEgZG8gUmVwb3NpdCZvYWN1dGU7cmlvIEluc3RpdHVjaW9uYWwgZGEgVW5pdmVyc2lkYWRlIEZlZGVyYWwgZGUgT3VybyBQcmV0bwogIDxicj4KICAoUkktVUZPUCksIG8gZGlyZWl0byBuJmF0aWxkZTtvLWV4Y2x1c2l2byBkZSByZXByb2R1emlyLCBjb252ZXJ0ZXIgKGNvbW8gZGVmaW5pZG8gYWJhaXhvKSBlL291IGRpc3RyaWJ1aXIgbyBkb2N1bWVudG8gZGVwb3NpdGFkbwogIDxicj4KICBlbSBmb3JtYXRvIGltcHJlc3NvLCBlbGV0ciZvY2lyYztuaWNvIG91IGVtIHF1YWxxdWVyIG91dHJvIG1laW8uCiAgPGJyPgogIDxicj4KICBWb2MmZWNpcmM7KHMpIGNvbmNvcmRhKG0pIHF1ZSBhIFVGT1AsIGdlc3RvcmEgZG8gUkktVUZPUCwgcG9kZSwgc2VtIGFsdGVyYXIgbyBjb250ZSZ1YWN1dGU7ZG8sIGNvbnZlcnRlciBvIGFycXVpdm8gZGVwb3NpdGFkbyBhCiAgPGJyPgogIHF1YWxxdWVyIG1laW8gb3UgZm9ybWF0byBjb20gZmlucyBkZSBwcmVzZXJ2YSZjY2VkaWw7JmF0aWxkZTtvLiBWb2MmZWNpcmM7KHMpIHRhbWImZWFjdXRlO20gY29uY29yZGEobSkgcXVlIGEgVUZPUCwgZ2VzdG9yYSBkbyBSSS1VRk9QLCBwb2RlCiAgPGJyPgogIG1hbnRlciBtYWlzIGRlIHVtYSBjJm9hY3V0ZTtwaWEgZGVzdGUgZGVwJm9hY3V0ZTtzaXRvIHBhcmEgZmlucyBkZSBzZWd1cmFuJmNjZWRpbDthLCA8ZW0+YmFjay11cDwvZW0+IGUvb3UgcHJlc2VydmEmY2NlZGlsOyZhdGlsZGU7by4KICA8YnI+CiAgPGJyPgogIFZvYyZlY2lyYzsocykgZGVjbGFyYShtKSBxdWUgYSBhcHJlc2VudGEmY2NlZGlsOyZhdGlsZGU7byBkbyBzZXUgdHJhYmFsaG8gJmVhY3V0ZTsgb3JpZ2luYWwgZSBxdWUgdm9jJmVjaXJjOyhzKSBwb2RlKG0pIGNvbmNlZGVyIG9zIGRpcmVpdG9zIGNvbnRpZG9zCiAgPGJyPgogIG5lc3RhIGxpY2VuJmNjZWRpbDthLiBWb2MmZWNpcmM7KHMpIHRhbWImZWFjdXRlO20gZGVjbGFyYShtKSBxdWUgbyBlbnZpbyAmZWFjdXRlOyBkZSBzZXUgY29uaGVjaW1lbnRvIGUgbiZhdGlsZGU7byBpbmZyaW5nZSBvcyBkaXJlaXRvcyBhdXRvcmFpcyBkZSBvdXRyYQogIDxicj4KICBwZXNzb2Egb3UgaW5zdGl0dWkmY2NlZGlsOyZhdGlsZGU7by4gQ2FzbyBvIGRvY3VtZW50byBhIHNlciBkZXBvc2l0YWRvIGNvbnRlbmhhIG1hdGVyaWFsIHBhcmEgbyBxdWFsIHZvYyZlY2lyYzsocykgbiZhdGlsZGU7byBkZXQmZWFjdXRlO20gYSB0aXR1bGFyaWRhZGUKICA8YnI+CiAgZG9zIGRpcmVpdG9zIGF1dG9yYWlzLCB2b2MmZWNpcmM7KHMpIGRlY2xhcmEobSkgcXVlIG9idGV2ZSBhIHBlcm1pc3MmYXRpbGRlO28gaXJyZXN0cml0YSBkbyB0aXR1bGFyIGRvcyBkaXJlaXRvcyBhdXRvcmFpcyBkZSBjb25jZWRlciAmYWdyYXZlOwogIDxicj4KICBVRk9QLCBnZXN0b3JhIGRvIFJJLVVGT1Agb3MgZGlyZWl0b3MgcmVxdWVyaWRvcyBwb3IgZXN0YSBsaWNlbiZjY2VkaWw7YSBlIHF1ZSBvcyBtYXRlcmlhaXMgZGUgcHJvcHJpZWRhZGUgZGUgdGVyY2Vpcm9zLCBlc3QmYXRpbGRlO28KICA8YnI+CiAgZGV2aWRhbWVudGUgaWRlbnRpZmljYWRvcyBlIHJlY29uaGVjaWRvcyBubyB0ZXh0byBvdSBjb250ZSZ1YWN1dGU7ZG8gZGEgYXByZXNlbnRhJmNjZWRpbDsmYXRpbGRlO28uCiAgPGJyPgogIDxicj4KICBDQVNPIE8gVFJBQkFMSE8gREVQT1NJVEFETyBURU5IQSBTSURPIEZJTkFOQ0lBRE8gT1UgQVBPSUFETyBQT1IgVU0gJk9hY3V0ZTtSRyZBdGlsZGU7TywgUVVFIE4mQXRpbGRlO08gQSBJTlNUSVRVSSZDY2VkaWw7JkF0aWxkZTtPIERFU1RFCiAgPGJyPgogIFJFU1BPU0lUJk9hY3V0ZTtSSU86IFZPQyZFY2lyYzsgREVDTEFSQSBURVIgQ1VNUFJJRE8gVE9ET1MgT1MgRElSRUlUT1MgREUgUkVWSVMmQXRpbGRlO08gRSBRVUFJU1FVRVIgT1VUUkFTIE9CUklHQSZDY2VkaWw7Jk90aWxkZTtFUwogIDxicj4KICBSRVFVRVJJREFTIFBFTE8gQ09OVFJBVE8gT1UgQUNPUkRPLiAKICA8YnI+CiAgPGJyPgogIE8gcmVwb3NpdCZvYWN1dGU7cmlvIGlkZW50aWZpY2FyJmFhY3V0ZTsgY2xhcmFtZW50ZSBvIHNldShzKSBub21lKHMpIGNvbW8gYXV0b3IoZXMpIG91IHRpdHVsYXIoZXMpIGRvIGRpcmVpdG8gZGUgYXV0b3IoZXMpIGRvIGRvY3VtZW50bwogIDxicj4KICBzdWJtZXRpZG8gZSBkZWNsYXJhIHF1ZSBuJmF0aWxkZTtvIGZhciZhYWN1dGU7IHF1YWxxdWVyIGFsdGVyYSZjY2VkaWw7JmF0aWxkZTtvIGFsJmVhY3V0ZTttIGRhcyBwZXJtaXRpZGFzIHBvciBlc3RhIGxpY2VuJmNjZWRpbDthLjwvcD4KPC9kaXY+CjwvYm9keT4KPC9odG1sPgo=Repositório InstitucionalPUBhttp://www.repositorio.ufop.br/oai/requestrepositorio@ufop.edu.bropendoar:32332019-06-10T18:34:58Repositório Institucional da UFOP - Universidade Federal de Ouro Preto (UFOP)false |
dc.title.pt_BR.fl_str_mv |
Development of a fluorescent based immunosensor for the serodiagnosis of canine Leishmaniasis combining immunomagnetic separation and flow cytometry. |
title |
Development of a fluorescent based immunosensor for the serodiagnosis of canine Leishmaniasis combining immunomagnetic separation and flow cytometry. |
spellingShingle |
Development of a fluorescent based immunosensor for the serodiagnosis of canine Leishmaniasis combining immunomagnetic separation and flow cytometry. Sousa, Susana Flow cytometry Canine leishmaniasis |
title_short |
Development of a fluorescent based immunosensor for the serodiagnosis of canine Leishmaniasis combining immunomagnetic separation and flow cytometry. |
title_full |
Development of a fluorescent based immunosensor for the serodiagnosis of canine Leishmaniasis combining immunomagnetic separation and flow cytometry. |
title_fullStr |
Development of a fluorescent based immunosensor for the serodiagnosis of canine Leishmaniasis combining immunomagnetic separation and flow cytometry. |
title_full_unstemmed |
Development of a fluorescent based immunosensor for the serodiagnosis of canine Leishmaniasis combining immunomagnetic separation and flow cytometry. |
title_sort |
Development of a fluorescent based immunosensor for the serodiagnosis of canine Leishmaniasis combining immunomagnetic separation and flow cytometry. |
author |
Sousa, Susana |
author_facet |
Sousa, Susana Cardoso, Luís Reed, Steven G. Reis, Alexandre Barbosa Martins Filho, Olindo Assis Silvestre, Ricardo Silva, Anabela Cordeiro da |
author_role |
author |
author2 |
Cardoso, Luís Reed, Steven G. Reis, Alexandre Barbosa Martins Filho, Olindo Assis Silvestre, Ricardo Silva, Anabela Cordeiro da |
author2_role |
author author author author author author |
dc.contributor.author.fl_str_mv |
Sousa, Susana Cardoso, Luís Reed, Steven G. Reis, Alexandre Barbosa Martins Filho, Olindo Assis Silvestre, Ricardo Silva, Anabela Cordeiro da |
dc.subject.por.fl_str_mv |
Flow cytometry Canine leishmaniasis |
topic |
Flow cytometry Canine leishmaniasis |
description |
An accurate diagnosis is essential for the control of infectious diseases. In the search for effective and efficient tests, biosensors have increasingly been exploited for the development of new and highly sensitive diagnostic methods. Here, we describe a new fluorescent based immunosensor comprising magnetic polymer microspheres coated with recombinant antigens to improve the detection of specific antibodies generated during an infectious disease. As a challenging model, we used canine leishmaniasis due to the unsatisfactory sensitivity associated with the detection of infection in asymptomatic animals where the levels of pathogen-specific antibodies are scarce. Methodology: Ni-NTA magnetic microspheres with 1,7 mm and 8,07 mm were coated with the Leishmania recombinant proteins LicTXNPx and rK39, respectively. A mixture of equal proportions of both recombinant protein-coated microspheres was used to recognize and specifically bind anti-rK39 and anti-LicTNXPx antibodies present in serum samples of infected dogs. The microspheres were recovered by magnetic separation and the percentage of fluorescent positive microspheres was quantified by flow cytometry. Principal Findings: A clinical evaluation carried out with 129 dog serum samples using the antigen combination demonstrated a sensitivity of 98,8% with a specificity of 94,4%. rK39 antigen alone demonstrated a higher sensitivity for symptomatic dogs (96,9%), while LicTXNPx antigen showed a higher sensitivity for asymptomatic (94,4%). Conclusions: Overall, our results demonstrated the potential of a magnetic microsphere associated flow cytometry methodology as a viable tool for highly sensitive laboratorial serodiagnosis of both clinical and subclinical forms of canine leishmaniasis. |
publishDate |
2013 |
dc.date.issued.fl_str_mv |
2013 |
dc.date.accessioned.fl_str_mv |
2015-01-19T16:42:44Z |
dc.date.available.fl_str_mv |
2015-01-19T16:42:44Z |
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.citation.fl_str_mv |
SOUSA, S. et al. Development of a fluorescent based immunosensor for the serodiagnosis of canine Leishmaniasis combining immunomagnetic separation and flow cytometry. PLoS Neglected Tropical Diseases, v. 7, p. e2371, 2013. Disponível em: <http://www.plosntds.org/article/info%3Adoi%2F10.1371%2Fjournal.pntd.0002371>. Aceso em: 12 ago. 2014. |
dc.identifier.uri.fl_str_mv |
http://www.repositorio.ufop.br/handle/123456789/4264 |
dc.identifier.issn.none.fl_str_mv |
1935-2735 |
dc.identifier.doi.none.fl_str_mv |
https://doi.org/10.1371/journal.pntd.0002371 |
identifier_str_mv |
SOUSA, S. et al. Development of a fluorescent based immunosensor for the serodiagnosis of canine Leishmaniasis combining immunomagnetic separation and flow cytometry. PLoS Neglected Tropical Diseases, v. 7, p. e2371, 2013. Disponível em: <http://www.plosntds.org/article/info%3Adoi%2F10.1371%2Fjournal.pntd.0002371>. Aceso em: 12 ago. 2014. 1935-2735 |
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http://www.repositorio.ufop.br/handle/123456789/4264 https://doi.org/10.1371/journal.pntd.0002371 |
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