Flow Cytometric microsphere-based immunoassay as a novel non-radiometric method for the detection of glutamic acid decarboxylase autoantibodies in type 1 Diabetes Mellitus
- Autores
- Guerra, Luciano Lucas; Trabucchi, Aldana; Faccinetti, Natalia Ines; Iacono, Ruben Francisco; Ureta, Daniela Beatriz; Poskus, Edgardo; Valdez, Silvina Noemi
- Año de publicación
- 2014
- Idioma
- inglés
- Tipo de recurso
- artículo
- Estado
- versión publicada
- Descripción
- The first measurable sign of arising autoimmunity in Type 1 Diabetes Mellitus is the detection of autoantibodies against beta-cell antigens, such as glutamic acid decarboxylase (GAD65). GAD65 autoantibodies (GADA) are usually measured by Radioligand Binding Assay (RBA). The aim of this work was to develop protocols of Flow Cytometric microsphere-based immunoassays (FloCMIA) which involved glutamic acid decarboxylase fused to thioredoxin (TrxGAD65) adsorbed on polystyrene microspheres. Detection of bound GADA was accomplished by the use of anti-human IgG-Alexa Fluor 488 (Protocol A), anti-human IgG-biotin and streptavidindichlorotriazinyl aminofluorescein (DTAF) (Protocol B) or TrxGAD65-biotin and streptavidin- DTAF (Protocol C). Serum samples obtained from 46 patients assayed for routine autoantibodies at Servicios Tecnológicos de Alto Nivel (STAN-CONICET) were analyzed by RBA, ELISA and three alternative FloCMIA designs. Protocol C exhibited the highest specificity (97.8%) and sensitivity (97.4%) and a wide dynamic range (1.00-134.40 SDs). Samples obtained from 40 new-onset diabetic patients were also analyzed to further evaluate the performance of protocol C. The latter protocol showed a sensitivity of 58.6% and a prevalence of 47.5%. Two patients resulted positive only by FloCMIA protocol C and its SDs were higher than RBA and ELISA, showing a significantly wide dynamic range. In conclusion, FloCMIA proved to be highly sensitive and specific, requiring a low sample volume; it is environmentally adequate, innovative and it represents a cost-effective alternative to traditional GADA determination by RBA and/or ELISA; making it applicable to most medium-complexity laboratories.
Fil: Guerra, Luciano Lucas. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral "Profesor R. A. Margni"; Argentina
Fil: Trabucchi, Aldana. Consejo Nacional de Investigaciones Cientiâficas y Tecnicas. Oficina de Coordinacion Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral "profesor R. A. Margni"; Argentina
Fil: Faccinetti, Natalia Ines. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral "Profesor R. A. Margni"; Argentina
Fil: Iacono, Ruben Francisco. Consejo Nacional de Investigaciones Cientiâficas y Tecnicas. Oficina de Coordinacion Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral "profesor R. A. Margni"; Argentina
Fil: Ureta, Daniela Beatriz. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral "Profesor R. A. Margni"; Argentina
Fil: Poskus, Edgardo. Consejo Nacional de Investigaciones Cientiâficas y Tecnicas. Oficina de Coordinacion Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral "profesor R. A. Margni"; Argentina
Fil: Valdez, Silvina Noemi. Consejo Nacional de Investigaciones Cientiâficas y Tecnicas. Oficina de Coordinacion Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral "profesor R. A. Margni"; Argentina - Materia
-
FLOW CYTOMETRY
DIABETES MELLITUS
GLUTAMIC ACID DECARBOXYLASE AUTOANTIBODIES
IMMUNOASSAY - Nivel de accesibilidad
- acceso abierto
- Condiciones de uso
- https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
- Repositorio
- Institución
- Consejo Nacional de Investigaciones Científicas y Técnicas
- OAI Identificador
- oai:ri.conicet.gov.ar:11336/8030
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oai:ri.conicet.gov.ar:11336/8030 |
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spelling |
Flow Cytometric microsphere-based immunoassay as a novel non-radiometric method for the detection of glutamic acid decarboxylase autoantibodies in type 1 Diabetes MellitusGuerra, Luciano LucasTrabucchi, AldanaFaccinetti, Natalia InesIacono, Ruben FranciscoUreta, Daniela BeatrizPoskus, EdgardoValdez, Silvina NoemiFLOW CYTOMETRYDIABETES MELLITUSGLUTAMIC ACID DECARBOXYLASE AUTOANTIBODIESIMMUNOASSAYhttps://purl.org/becyt/ford/3.4https://purl.org/becyt/ford/3The first measurable sign of arising autoimmunity in Type 1 Diabetes Mellitus is the detection of autoantibodies against beta-cell antigens, such as glutamic acid decarboxylase (GAD65). GAD65 autoantibodies (GADA) are usually measured by Radioligand Binding Assay (RBA). The aim of this work was to develop protocols of Flow Cytometric microsphere-based immunoassays (FloCMIA) which involved glutamic acid decarboxylase fused to thioredoxin (TrxGAD65) adsorbed on polystyrene microspheres. Detection of bound GADA was accomplished by the use of anti-human IgG-Alexa Fluor 488 (Protocol A), anti-human IgG-biotin and streptavidindichlorotriazinyl aminofluorescein (DTAF) (Protocol B) or TrxGAD65-biotin and streptavidin- DTAF (Protocol C). Serum samples obtained from 46 patients assayed for routine autoantibodies at Servicios Tecnológicos de Alto Nivel (STAN-CONICET) were analyzed by RBA, ELISA and three alternative FloCMIA designs. Protocol C exhibited the highest specificity (97.8%) and sensitivity (97.4%) and a wide dynamic range (1.00-134.40 SDs). Samples obtained from 40 new-onset diabetic patients were also analyzed to further evaluate the performance of protocol C. The latter protocol showed a sensitivity of 58.6% and a prevalence of 47.5%. Two patients resulted positive only by FloCMIA protocol C and its SDs were higher than RBA and ELISA, showing a significantly wide dynamic range. In conclusion, FloCMIA proved to be highly sensitive and specific, requiring a low sample volume; it is environmentally adequate, innovative and it represents a cost-effective alternative to traditional GADA determination by RBA and/or ELISA; making it applicable to most medium-complexity laboratories.Fil: Guerra, Luciano Lucas. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral "Profesor R. A. Margni"; ArgentinaFil: Trabucchi, Aldana. Consejo Nacional de Investigaciones Cientiâficas y Tecnicas. Oficina de Coordinacion Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral "profesor R. A. Margni"; ArgentinaFil: Faccinetti, Natalia Ines. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral "Profesor R. A. Margni"; ArgentinaFil: Iacono, Ruben Francisco. Consejo Nacional de Investigaciones Cientiâficas y Tecnicas. Oficina de Coordinacion Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral "profesor R. A. Margni"; ArgentinaFil: Ureta, Daniela Beatriz. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral "Profesor R. A. Margni"; ArgentinaFil: Poskus, Edgardo. Consejo Nacional de Investigaciones Cientiâficas y Tecnicas. Oficina de Coordinacion Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral "profesor R. A. Margni"; ArgentinaFil: Valdez, Silvina Noemi. Consejo Nacional de Investigaciones Cientiâficas y Tecnicas. Oficina de Coordinacion Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral "profesor R. A. Margni"; ArgentinaRoyal Society Of Chemistry2014-02info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfapplication/pdfapplication/pdfapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/8030Guerra, Luciano Lucas; Trabucchi, Aldana; Faccinetti, Natalia Ines; Iacono, Ruben Francisco; Ureta, Daniela Beatriz; et al.; Flow Cytometric microsphere-based immunoassay as a novel non-radiometric method for the detection of glutamic acid decarboxylase autoantibodies in type 1 Diabetes Mellitus; Royal Society Of Chemistry; Analyst; 139; 12; 2-2014; 3017-30250003-2654enginfo:eu-repo/semantics/altIdentifier/url/http://pubs.rsc.org/en/Content/ArticleLanding/2014/AN/c3an02243a#!divAbstractinfo:eu-repo/semantics/altIdentifier/doi/10.1039/C3AN02243Ainfo:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by-nc-sa/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-09-03T10:05:47Zoai:ri.conicet.gov.ar:11336/8030instacron:CONICETInstitucionalhttp://ri.conicet.gov.ar/Organismo científico-tecnológicoNo correspondehttp://ri.conicet.gov.ar/oai/requestdasensio@conicet.gov.ar; lcarlino@conicet.gov.arArgentinaNo correspondeNo correspondeNo correspondeopendoar:34982025-09-03 10:05:47.448CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse |
dc.title.none.fl_str_mv |
Flow Cytometric microsphere-based immunoassay as a novel non-radiometric method for the detection of glutamic acid decarboxylase autoantibodies in type 1 Diabetes Mellitus |
title |
Flow Cytometric microsphere-based immunoassay as a novel non-radiometric method for the detection of glutamic acid decarboxylase autoantibodies in type 1 Diabetes Mellitus |
spellingShingle |
Flow Cytometric microsphere-based immunoassay as a novel non-radiometric method for the detection of glutamic acid decarboxylase autoantibodies in type 1 Diabetes Mellitus Guerra, Luciano Lucas FLOW CYTOMETRY DIABETES MELLITUS GLUTAMIC ACID DECARBOXYLASE AUTOANTIBODIES IMMUNOASSAY |
title_short |
Flow Cytometric microsphere-based immunoassay as a novel non-radiometric method for the detection of glutamic acid decarboxylase autoantibodies in type 1 Diabetes Mellitus |
title_full |
Flow Cytometric microsphere-based immunoassay as a novel non-radiometric method for the detection of glutamic acid decarboxylase autoantibodies in type 1 Diabetes Mellitus |
title_fullStr |
Flow Cytometric microsphere-based immunoassay as a novel non-radiometric method for the detection of glutamic acid decarboxylase autoantibodies in type 1 Diabetes Mellitus |
title_full_unstemmed |
Flow Cytometric microsphere-based immunoassay as a novel non-radiometric method for the detection of glutamic acid decarboxylase autoantibodies in type 1 Diabetes Mellitus |
title_sort |
Flow Cytometric microsphere-based immunoassay as a novel non-radiometric method for the detection of glutamic acid decarboxylase autoantibodies in type 1 Diabetes Mellitus |
dc.creator.none.fl_str_mv |
Guerra, Luciano Lucas Trabucchi, Aldana Faccinetti, Natalia Ines Iacono, Ruben Francisco Ureta, Daniela Beatriz Poskus, Edgardo Valdez, Silvina Noemi |
author |
Guerra, Luciano Lucas |
author_facet |
Guerra, Luciano Lucas Trabucchi, Aldana Faccinetti, Natalia Ines Iacono, Ruben Francisco Ureta, Daniela Beatriz Poskus, Edgardo Valdez, Silvina Noemi |
author_role |
author |
author2 |
Trabucchi, Aldana Faccinetti, Natalia Ines Iacono, Ruben Francisco Ureta, Daniela Beatriz Poskus, Edgardo Valdez, Silvina Noemi |
author2_role |
author author author author author author |
dc.subject.none.fl_str_mv |
FLOW CYTOMETRY DIABETES MELLITUS GLUTAMIC ACID DECARBOXYLASE AUTOANTIBODIES IMMUNOASSAY |
topic |
FLOW CYTOMETRY DIABETES MELLITUS GLUTAMIC ACID DECARBOXYLASE AUTOANTIBODIES IMMUNOASSAY |
purl_subject.fl_str_mv |
https://purl.org/becyt/ford/3.4 https://purl.org/becyt/ford/3 |
dc.description.none.fl_txt_mv |
The first measurable sign of arising autoimmunity in Type 1 Diabetes Mellitus is the detection of autoantibodies against beta-cell antigens, such as glutamic acid decarboxylase (GAD65). GAD65 autoantibodies (GADA) are usually measured by Radioligand Binding Assay (RBA). The aim of this work was to develop protocols of Flow Cytometric microsphere-based immunoassays (FloCMIA) which involved glutamic acid decarboxylase fused to thioredoxin (TrxGAD65) adsorbed on polystyrene microspheres. Detection of bound GADA was accomplished by the use of anti-human IgG-Alexa Fluor 488 (Protocol A), anti-human IgG-biotin and streptavidindichlorotriazinyl aminofluorescein (DTAF) (Protocol B) or TrxGAD65-biotin and streptavidin- DTAF (Protocol C). Serum samples obtained from 46 patients assayed for routine autoantibodies at Servicios Tecnológicos de Alto Nivel (STAN-CONICET) were analyzed by RBA, ELISA and three alternative FloCMIA designs. Protocol C exhibited the highest specificity (97.8%) and sensitivity (97.4%) and a wide dynamic range (1.00-134.40 SDs). Samples obtained from 40 new-onset diabetic patients were also analyzed to further evaluate the performance of protocol C. The latter protocol showed a sensitivity of 58.6% and a prevalence of 47.5%. Two patients resulted positive only by FloCMIA protocol C and its SDs were higher than RBA and ELISA, showing a significantly wide dynamic range. In conclusion, FloCMIA proved to be highly sensitive and specific, requiring a low sample volume; it is environmentally adequate, innovative and it represents a cost-effective alternative to traditional GADA determination by RBA and/or ELISA; making it applicable to most medium-complexity laboratories. Fil: Guerra, Luciano Lucas. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral "Profesor R. A. Margni"; Argentina Fil: Trabucchi, Aldana. Consejo Nacional de Investigaciones Cientiâficas y Tecnicas. Oficina de Coordinacion Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral "profesor R. A. Margni"; Argentina Fil: Faccinetti, Natalia Ines. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral "Profesor R. A. Margni"; Argentina Fil: Iacono, Ruben Francisco. Consejo Nacional de Investigaciones Cientiâficas y Tecnicas. Oficina de Coordinacion Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral "profesor R. A. Margni"; Argentina Fil: Ureta, Daniela Beatriz. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral "Profesor R. A. Margni"; Argentina Fil: Poskus, Edgardo. Consejo Nacional de Investigaciones Cientiâficas y Tecnicas. Oficina de Coordinacion Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral "profesor R. A. Margni"; Argentina Fil: Valdez, Silvina Noemi. Consejo Nacional de Investigaciones Cientiâficas y Tecnicas. Oficina de Coordinacion Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral "profesor R. A. Margni"; Argentina |
description |
The first measurable sign of arising autoimmunity in Type 1 Diabetes Mellitus is the detection of autoantibodies against beta-cell antigens, such as glutamic acid decarboxylase (GAD65). GAD65 autoantibodies (GADA) are usually measured by Radioligand Binding Assay (RBA). The aim of this work was to develop protocols of Flow Cytometric microsphere-based immunoassays (FloCMIA) which involved glutamic acid decarboxylase fused to thioredoxin (TrxGAD65) adsorbed on polystyrene microspheres. Detection of bound GADA was accomplished by the use of anti-human IgG-Alexa Fluor 488 (Protocol A), anti-human IgG-biotin and streptavidindichlorotriazinyl aminofluorescein (DTAF) (Protocol B) or TrxGAD65-biotin and streptavidin- DTAF (Protocol C). Serum samples obtained from 46 patients assayed for routine autoantibodies at Servicios Tecnológicos de Alto Nivel (STAN-CONICET) were analyzed by RBA, ELISA and three alternative FloCMIA designs. Protocol C exhibited the highest specificity (97.8%) and sensitivity (97.4%) and a wide dynamic range (1.00-134.40 SDs). Samples obtained from 40 new-onset diabetic patients were also analyzed to further evaluate the performance of protocol C. The latter protocol showed a sensitivity of 58.6% and a prevalence of 47.5%. Two patients resulted positive only by FloCMIA protocol C and its SDs were higher than RBA and ELISA, showing a significantly wide dynamic range. In conclusion, FloCMIA proved to be highly sensitive and specific, requiring a low sample volume; it is environmentally adequate, innovative and it represents a cost-effective alternative to traditional GADA determination by RBA and/or ELISA; making it applicable to most medium-complexity laboratories. |
publishDate |
2014 |
dc.date.none.fl_str_mv |
2014-02 |
dc.type.none.fl_str_mv |
info:eu-repo/semantics/article info:eu-repo/semantics/publishedVersion http://purl.org/coar/resource_type/c_6501 info:ar-repo/semantics/articulo |
format |
article |
status_str |
publishedVersion |
dc.identifier.none.fl_str_mv |
http://hdl.handle.net/11336/8030 Guerra, Luciano Lucas; Trabucchi, Aldana; Faccinetti, Natalia Ines; Iacono, Ruben Francisco; Ureta, Daniela Beatriz; et al.; Flow Cytometric microsphere-based immunoassay as a novel non-radiometric method for the detection of glutamic acid decarboxylase autoantibodies in type 1 Diabetes Mellitus; Royal Society Of Chemistry; Analyst; 139; 12; 2-2014; 3017-3025 0003-2654 |
url |
http://hdl.handle.net/11336/8030 |
identifier_str_mv |
Guerra, Luciano Lucas; Trabucchi, Aldana; Faccinetti, Natalia Ines; Iacono, Ruben Francisco; Ureta, Daniela Beatriz; et al.; Flow Cytometric microsphere-based immunoassay as a novel non-radiometric method for the detection of glutamic acid decarboxylase autoantibodies in type 1 Diabetes Mellitus; Royal Society Of Chemistry; Analyst; 139; 12; 2-2014; 3017-3025 0003-2654 |
dc.language.none.fl_str_mv |
eng |
language |
eng |
dc.relation.none.fl_str_mv |
info:eu-repo/semantics/altIdentifier/url/http://pubs.rsc.org/en/Content/ArticleLanding/2014/AN/c3an02243a#!divAbstract info:eu-repo/semantics/altIdentifier/doi/10.1039/C3AN02243A |
dc.rights.none.fl_str_mv |
info:eu-repo/semantics/openAccess https://creativecommons.org/licenses/by-nc-sa/2.5/ar/ |
eu_rights_str_mv |
openAccess |
rights_invalid_str_mv |
https://creativecommons.org/licenses/by-nc-sa/2.5/ar/ |
dc.format.none.fl_str_mv |
application/pdf application/pdf application/pdf application/pdf application/pdf |
dc.publisher.none.fl_str_mv |
Royal Society Of Chemistry |
publisher.none.fl_str_mv |
Royal Society Of Chemistry |
dc.source.none.fl_str_mv |
reponame:CONICET Digital (CONICET) instname:Consejo Nacional de Investigaciones Científicas y Técnicas |
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CONICET Digital (CONICET) |
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CONICET Digital (CONICET) |
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Consejo Nacional de Investigaciones Científicas y Técnicas |
repository.name.fl_str_mv |
CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicas |
repository.mail.fl_str_mv |
dasensio@conicet.gov.ar; lcarlino@conicet.gov.ar |
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13.13397 |