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Artículo

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 LucasIcon ; Trabucchi, AldanaIcon ; Faccinetti, Natalia InesIcon ; Iacono, Ruben FranciscoIcon ; Ureta, Daniela BeatrizIcon ; Poskus, EdgardoIcon ; Valdez, Silvina NoemiIcon
Fecha de publicación: 02/2014
Editorial: Royal Society Of Chemistry
Revista: Analyst
ISSN: 0003-2654
Idioma: Inglés
Tipo de recurso: Artículo publicado
Clasificación temática:
Biotecnología relacionada con la Salud

Resumen

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.
Palabras clave: Flow Cytometry , Diabetes Mellitus , Glutamic Acid Decarboxylase Autoantibodies , Immunoassay
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info:eu-repo/semantics/openAccess Excepto donde se diga explícitamente, este item se publica bajo la siguiente descripción: Creative Commons Attribution-NonCommercial-ShareAlike 2.5 Unported (CC BY-NC-SA 2.5)
Identificadores
URI: http://hdl.handle.net/11336/8030
URL: http://pubs.rsc.org/en/Content/ArticleLanding/2014/AN/c3an02243a#!divAbstract
DOI: http://dx.doi.org/10.1039/C3AN02243A
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Articulos(IDEHU) [207]
Articulos de INST.DE EST.DE LA INMUNIDAD HUMORAL PROF.R.A.MARGNI
Citación
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
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