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
CONICET Digital (CONICET)
Institución
Consejo Nacional de Investigaciones Científicas y Técnicas
OAI Identificador
oai:ri.conicet.gov.ar:11336/8030
Acceder
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oai_identifier_str oai:ri.conicet.gov.ar:11336/8030
network_acronym_str CONICETDig
repository_id_str 3498
network_name_str CONICET Digital (CONICET)
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 Técnicas2025-09-29T10:27:51Zoai: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:34982025-09-29 10:27:52.271CONICET 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
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info:eu-repo/semantics/altIdentifier/doi/10.1039/C3AN02243A
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
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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
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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)
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reponame_str CONICET Digital (CONICET)
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instname_str Consejo Nacional de Investigaciones Científicas y Técnicas
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repository.mail.fl_str_mv dasensio@conicet.gov.ar; lcarlino@conicet.gov.ar
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