Seleção, caracterização e aplicação de anticorpos scFv (single chain variable fragment) na captura de antígenos para o sorodiagnóstico da neurocisticercose humana
Autor(a) principal: | |
---|---|
Data de Publicação: | 2012 |
Tipo de documento: | Tese |
Idioma: | por |
Título da fonte: | Repositório Institucional da UFU |
Texto Completo: | https://repositorio.ufu.br/handle/123456789/16574 https://doi.org/10.14393/ufu.te.2012.59 |
Resumo: | Human neurocysticercosis (NC) is an important but neglected cause of epilepsy in developing countries where the parasite occurs. Expression of single-chain variable fragment (scFv) antibodies on the surface of bacteriophage is widely used to prepare antibodies with pre-defined specificities. A phage antibody library was selected against peptides displayed on phages coupled to beads and total saline extract of Taenia solium metacestodes immobilized on microtiter plate wells. After two rounds of selection 96 phage clones of each panning were selected, tested for scFv expression and specificity to each target. Specific clones were further analyzed by ELISA (Enzyme-linked immunosorbent assay), Dot-blot, sequencing and immunofluorescence. After selection, three clones were used for antigen capture to characterize its targets for future immunodiagnostic assays development. Total saline extract was fractionated on ion exchange resin diethylaminoethyl (DEAE), and fractions were tested by ELISA to detect sera IgG from: NC, other parasites and health controls (40 each). The fractions with best diagnostic parameters (sensitivity, specificity, area under curve and likelihood ratio, calculated by TG-ROC) were selected and subjected to antigen capture using each purified scFv clone. Each captured fraction was tested by ELISA to detect IgG in 30 serum samples from each group. In immunofluorescence tests, no fluorescence was observed in negative controls, and all clones showed a non-uniform staining profile, and their targets were elucidated through mass spectrometry. After ion exchange fractionation and ELISA tests, DEAE S2 fraction showed to be the best one and was used to capture new antigens. DEAE S2 showed 93.3% specificity. Among all clones, A4 and B6 captured antigens from saline extract and DEAE S2 fraction, respectively, with the best diagnostic parameters. In conclusion, antibody phage display technology is a potential approach for the study of antigen-antibody interactions, which can be used to further elucidate the biology of interaction on neurocysticercosis and to capture new antigens with potential applications in NC diagnosis and therapeutics. |
id |
UFU_455a420de034c7faba9a49bd8e8a50bc |
---|---|
oai_identifier_str |
oai:repositorio.ufu.br:123456789/16574 |
network_acronym_str |
UFU |
network_name_str |
Repositório Institucional da UFU |
repository_id_str |
|
spelling |
2016-06-22T18:46:19Z2012-09-142016-06-22T18:46:19Z2012-07-06RIBEIRO, Vanessa da Silva. Selection, characterization and aplication of scFv antibodies (single chain variable fragment) to capture antigens for human neurocysticercosis serodiagnosis. 2012. 89 f. Tese (Doutorado em Ciências Biológicas) - Universidade Federal de Uberlândia, Uberlândia, 2012. DOI https://doi.org/10.14393/ufu.te.2012.59https://repositorio.ufu.br/handle/123456789/16574https://doi.org/10.14393/ufu.te.2012.59Human neurocysticercosis (NC) is an important but neglected cause of epilepsy in developing countries where the parasite occurs. Expression of single-chain variable fragment (scFv) antibodies on the surface of bacteriophage is widely used to prepare antibodies with pre-defined specificities. A phage antibody library was selected against peptides displayed on phages coupled to beads and total saline extract of Taenia solium metacestodes immobilized on microtiter plate wells. After two rounds of selection 96 phage clones of each panning were selected, tested for scFv expression and specificity to each target. Specific clones were further analyzed by ELISA (Enzyme-linked immunosorbent assay), Dot-blot, sequencing and immunofluorescence. After selection, three clones were used for antigen capture to characterize its targets for future immunodiagnostic assays development. Total saline extract was fractionated on ion exchange resin diethylaminoethyl (DEAE), and fractions were tested by ELISA to detect sera IgG from: NC, other parasites and health controls (40 each). The fractions with best diagnostic parameters (sensitivity, specificity, area under curve and likelihood ratio, calculated by TG-ROC) were selected and subjected to antigen capture using each purified scFv clone. Each captured fraction was tested by ELISA to detect IgG in 30 serum samples from each group. In immunofluorescence tests, no fluorescence was observed in negative controls, and all clones showed a non-uniform staining profile, and their targets were elucidated through mass spectrometry. After ion exchange fractionation and ELISA tests, DEAE S2 fraction showed to be the best one and was used to capture new antigens. DEAE S2 showed 93.3% specificity. Among all clones, A4 and B6 captured antigens from saline extract and DEAE S2 fraction, respectively, with the best diagnostic parameters. In conclusion, antibody phage display technology is a potential approach for the study of antigen-antibody interactions, which can be used to further elucidate the biology of interaction on neurocysticercosis and to capture new antigens with potential applications in NC diagnosis and therapeutics.A neurocistocercose humana (NC) é uma doença muito importante, porém negligenciada e é a maior causa de epilepsia em países em desenvolvimento onde a parasitose ocorre. A expressão de fragmentos de cadeia única das regiões variáveis de anticorpo (scFv) na superfície de bacteriófagos é amplamente utilizada para obter anticorpos com especificidades pré-definidas. Uma biblioteca de anticorpos foi utilizada para a seleção de clones específicos à peptídeos expostos em fagos acoplados a beads e ao extrato salino total de Taenia solium (S) imobilizado em placas de microtitulação. Após dois ciclos de seleção, 96 clones de anticorpos foram selecionados contra cada alvo, testados para expressão do scFv e especificidade pelo alvo. Aqueles clones que se mostraram específicos foram melhor analisados por ELISA (Enzyme linked immunosorbent assay), Dot blot, sequenciamento e imunofluorescência. Três clones foram selecionados para serem utilizados na captura antigênica e caracterização do antígeno verdadeiro e para captura de novos antígenos com potencial aplicação em testes diagnósticos. O extrato S foi fracionado em resina de troca iônica diethylaminoethyl (DEAE) para obter frações que foram posteriormente testadas por ELISA para detectar IgG em amostras de soro de pacientes: com NC, outras parasitoses e saudáveis, 40 amostras cada grupo. A fração com melhores parâmetros diagnósticos (sensibilidade, especificidade, área sob a curva e likelihood ratio, calculadas por TG-ROC) foi selecionada e sujeita à captura antigênica usando cada clone de scFv purificado. Cada fração capturada foi testada por ELISA para detectar IgG em 30 amostras de soro de cada grupo. Nos testes de imunofluorescência, nenhuma fluorescência foi observada com os controles negativos e todos os clones mostraram um padrão de marcação não uniforme, seus antígenos alvo foram elucidados por espectrometria de massas. Após fracionamento por troca iônica e ELISA, a fração DEAE S2 se mostrou a melhor e foi utilizada para a captura de novos antígenos. A fração DEAE S2 mostrou especificidade de 93,3%. Dentre todos os clones, o A4 e o B6 capturaram antígenos do extrato S e fração DEAE S2, respectivamente, com os melhores parâmetros diagnósticos. Em conclusão a tecnologia de exposição de anticorpos em fagos é uma técnica potencial para o estudo de interações antígeno-anticorpo utilizadas para melhor elucidar a a biologia da interação na NC e para capturar novos antígenos potencialmente aplicáveis para o diagnóstico da NC.Doutor em Imunologia e Parasitologia Aplicadasapplication/pdfporUniversidade Federal de UberlândiaPrograma de Pós-graduação em Imunologia e Parasitologia AplicadasUFUBRCiências BiológicasNeurocisticercoseAnticorpos scFvTaenia soliumDiagnósticoCisticercose cerebrospinalAnticorposNeurocysticercosisscFv antibodiesPhage displayDiagnosisCNPQ::CIENCIAS BIOLOGICAS::PARASITOLOGIASeleção, caracterização e aplicação de anticorpos scFv (single chain variable fragment) na captura de antígenos para o sorodiagnóstico da neurocisticercose humanaSelection, characterization and aplication of scFv antibodies (single chain variable fragment) to capture antigens for human neurocysticercosis serodiagnosisinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/doctoralThesisGoulart Filho, Luiz Ricardohttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4781012P8Costa-Cruz, Julia Mariahttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4797155D9Souza, Guilherme Rocha Lino dehttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4700389E3Cunha Junior, Jair Pereira dahttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4795802Y5Oliveira, Heliana Batista dehttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4705324P3Cardoso, Ronehttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4764938H3http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4162741U8Ribeiro, Vanessa da Silva81761667info:eu-repo/semantics/openAccessreponame:Repositório Institucional da UFUinstname:Universidade Federal de Uberlândia (UFU)instacron:UFUTHUMBNAILt.pdf.jpgt.pdf.jpgGenerated Thumbnailimage/jpeg1333https://repositorio.ufu.br/bitstream/123456789/16574/3/t.pdf.jpg1a3de6e4d647d9f3df6603a94a2245d5MD53ORIGINALt.pdfapplication/pdf2727113https://repositorio.ufu.br/bitstream/123456789/16574/1/t.pdf43bf7f0f05c2c9a97fffa74fc8f68165MD51TEXTt.pdf.txtt.pdf.txtExtracted texttext/plain173755https://repositorio.ufu.br/bitstream/123456789/16574/2/t.pdf.txt4e6e21c8d0f11f0d30e22d62ba454a01MD52123456789/165742022-09-29 13:36:37.22oai:repositorio.ufu.br:123456789/16574Repositório InstitucionalONGhttp://repositorio.ufu.br/oai/requestdiinf@dirbi.ufu.bropendoar:2022-09-29T16:36:37Repositório Institucional da UFU - Universidade Federal de Uberlândia (UFU)false |
dc.title.por.fl_str_mv |
Seleção, caracterização e aplicação de anticorpos scFv (single chain variable fragment) na captura de antígenos para o sorodiagnóstico da neurocisticercose humana |
dc.title.alternative.eng.fl_str_mv |
Selection, characterization and aplication of scFv antibodies (single chain variable fragment) to capture antigens for human neurocysticercosis serodiagnosis |
title |
Seleção, caracterização e aplicação de anticorpos scFv (single chain variable fragment) na captura de antígenos para o sorodiagnóstico da neurocisticercose humana |
spellingShingle |
Seleção, caracterização e aplicação de anticorpos scFv (single chain variable fragment) na captura de antígenos para o sorodiagnóstico da neurocisticercose humana Ribeiro, Vanessa da Silva Neurocisticercose Anticorpos scFv Taenia solium Diagnóstico Cisticercose cerebrospinal Anticorpos Neurocysticercosis scFv antibodies Phage display Diagnosis CNPQ::CIENCIAS BIOLOGICAS::PARASITOLOGIA |
title_short |
Seleção, caracterização e aplicação de anticorpos scFv (single chain variable fragment) na captura de antígenos para o sorodiagnóstico da neurocisticercose humana |
title_full |
Seleção, caracterização e aplicação de anticorpos scFv (single chain variable fragment) na captura de antígenos para o sorodiagnóstico da neurocisticercose humana |
title_fullStr |
Seleção, caracterização e aplicação de anticorpos scFv (single chain variable fragment) na captura de antígenos para o sorodiagnóstico da neurocisticercose humana |
title_full_unstemmed |
Seleção, caracterização e aplicação de anticorpos scFv (single chain variable fragment) na captura de antígenos para o sorodiagnóstico da neurocisticercose humana |
title_sort |
Seleção, caracterização e aplicação de anticorpos scFv (single chain variable fragment) na captura de antígenos para o sorodiagnóstico da neurocisticercose humana |
author |
Ribeiro, Vanessa da Silva |
author_facet |
Ribeiro, Vanessa da Silva |
author_role |
author |
dc.contributor.advisor-co1.fl_str_mv |
Goulart Filho, Luiz Ricardo |
dc.contributor.advisor-co1Lattes.fl_str_mv |
http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4781012P8 |
dc.contributor.advisor1.fl_str_mv |
Costa-Cruz, Julia Maria |
dc.contributor.advisor1Lattes.fl_str_mv |
http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4797155D9 |
dc.contributor.referee1.fl_str_mv |
Souza, Guilherme Rocha Lino de |
dc.contributor.referee1Lattes.fl_str_mv |
http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4700389E3 |
dc.contributor.referee2.fl_str_mv |
Cunha Junior, Jair Pereira da |
dc.contributor.referee2Lattes.fl_str_mv |
http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4795802Y5 |
dc.contributor.referee3.fl_str_mv |
Oliveira, Heliana Batista de |
dc.contributor.referee3Lattes.fl_str_mv |
http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4705324P3 |
dc.contributor.referee4.fl_str_mv |
Cardoso, Rone |
dc.contributor.referee4Lattes.fl_str_mv |
http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4764938H3 |
dc.contributor.authorLattes.fl_str_mv |
http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4162741U8 |
dc.contributor.author.fl_str_mv |
Ribeiro, Vanessa da Silva |
contributor_str_mv |
Goulart Filho, Luiz Ricardo Costa-Cruz, Julia Maria Souza, Guilherme Rocha Lino de Cunha Junior, Jair Pereira da Oliveira, Heliana Batista de Cardoso, Rone |
dc.subject.por.fl_str_mv |
Neurocisticercose Anticorpos scFv Taenia solium Diagnóstico Cisticercose cerebrospinal Anticorpos |
topic |
Neurocisticercose Anticorpos scFv Taenia solium Diagnóstico Cisticercose cerebrospinal Anticorpos Neurocysticercosis scFv antibodies Phage display Diagnosis CNPQ::CIENCIAS BIOLOGICAS::PARASITOLOGIA |
dc.subject.eng.fl_str_mv |
Neurocysticercosis scFv antibodies Phage display Diagnosis |
dc.subject.cnpq.fl_str_mv |
CNPQ::CIENCIAS BIOLOGICAS::PARASITOLOGIA |
description |
Human neurocysticercosis (NC) is an important but neglected cause of epilepsy in developing countries where the parasite occurs. Expression of single-chain variable fragment (scFv) antibodies on the surface of bacteriophage is widely used to prepare antibodies with pre-defined specificities. A phage antibody library was selected against peptides displayed on phages coupled to beads and total saline extract of Taenia solium metacestodes immobilized on microtiter plate wells. After two rounds of selection 96 phage clones of each panning were selected, tested for scFv expression and specificity to each target. Specific clones were further analyzed by ELISA (Enzyme-linked immunosorbent assay), Dot-blot, sequencing and immunofluorescence. After selection, three clones were used for antigen capture to characterize its targets for future immunodiagnostic assays development. Total saline extract was fractionated on ion exchange resin diethylaminoethyl (DEAE), and fractions were tested by ELISA to detect sera IgG from: NC, other parasites and health controls (40 each). The fractions with best diagnostic parameters (sensitivity, specificity, area under curve and likelihood ratio, calculated by TG-ROC) were selected and subjected to antigen capture using each purified scFv clone. Each captured fraction was tested by ELISA to detect IgG in 30 serum samples from each group. In immunofluorescence tests, no fluorescence was observed in negative controls, and all clones showed a non-uniform staining profile, and their targets were elucidated through mass spectrometry. After ion exchange fractionation and ELISA tests, DEAE S2 fraction showed to be the best one and was used to capture new antigens. DEAE S2 showed 93.3% specificity. Among all clones, A4 and B6 captured antigens from saline extract and DEAE S2 fraction, respectively, with the best diagnostic parameters. In conclusion, antibody phage display technology is a potential approach for the study of antigen-antibody interactions, which can be used to further elucidate the biology of interaction on neurocysticercosis and to capture new antigens with potential applications in NC diagnosis and therapeutics. |
publishDate |
2012 |
dc.date.available.fl_str_mv |
2012-09-14 2016-06-22T18:46:19Z |
dc.date.issued.fl_str_mv |
2012-07-06 |
dc.date.accessioned.fl_str_mv |
2016-06-22T18:46:19Z |
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 |
RIBEIRO, Vanessa da Silva. Selection, characterization and aplication of scFv antibodies (single chain variable fragment) to capture antigens for human neurocysticercosis serodiagnosis. 2012. 89 f. Tese (Doutorado em Ciências Biológicas) - Universidade Federal de Uberlândia, Uberlândia, 2012. DOI https://doi.org/10.14393/ufu.te.2012.59 |
dc.identifier.uri.fl_str_mv |
https://repositorio.ufu.br/handle/123456789/16574 |
dc.identifier.doi.none.fl_str_mv |
https://doi.org/10.14393/ufu.te.2012.59 |
identifier_str_mv |
RIBEIRO, Vanessa da Silva. Selection, characterization and aplication of scFv antibodies (single chain variable fragment) to capture antigens for human neurocysticercosis serodiagnosis. 2012. 89 f. Tese (Doutorado em Ciências Biológicas) - Universidade Federal de Uberlândia, Uberlândia, 2012. DOI https://doi.org/10.14393/ufu.te.2012.59 |
url |
https://repositorio.ufu.br/handle/123456789/16574 https://doi.org/10.14393/ufu.te.2012.59 |
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 Uberlândia |
dc.publisher.program.fl_str_mv |
Programa de Pós-graduação em Imunologia e Parasitologia Aplicadas |
dc.publisher.initials.fl_str_mv |
UFU |
dc.publisher.country.fl_str_mv |
BR |
dc.publisher.department.fl_str_mv |
Ciências Biológicas |
publisher.none.fl_str_mv |
Universidade Federal de Uberlândia |
dc.source.none.fl_str_mv |
reponame:Repositório Institucional da UFU instname:Universidade Federal de Uberlândia (UFU) instacron:UFU |
instname_str |
Universidade Federal de Uberlândia (UFU) |
instacron_str |
UFU |
institution |
UFU |
reponame_str |
Repositório Institucional da UFU |
collection |
Repositório Institucional da UFU |
bitstream.url.fl_str_mv |
https://repositorio.ufu.br/bitstream/123456789/16574/3/t.pdf.jpg https://repositorio.ufu.br/bitstream/123456789/16574/1/t.pdf https://repositorio.ufu.br/bitstream/123456789/16574/2/t.pdf.txt |
bitstream.checksum.fl_str_mv |
1a3de6e4d647d9f3df6603a94a2245d5 43bf7f0f05c2c9a97fffa74fc8f68165 4e6e21c8d0f11f0d30e22d62ba454a01 |
bitstream.checksumAlgorithm.fl_str_mv |
MD5 MD5 MD5 |
repository.name.fl_str_mv |
Repositório Institucional da UFU - Universidade Federal de Uberlândia (UFU) |
repository.mail.fl_str_mv |
diinf@dirbi.ufu.br |
_version_ |
1802110339010002944 |