IDEHU   05542
INSTITUTO DE ESTUDIOS DE LA INMUNIDAD HUMORAL PROF. RICARDO A. MARGNI
Unidad Ejecutora - UE
artículos
Título:
Flow Cytometric microsphere-based immunoassay as a novel non-radiometric method for the detection of glutamic acid decarboxylase autoantibodies in type 1 Diabetes Mellitus
Autor/es:
GUERRA, LUCIANO; TRABUCCHI ALDANA; FACCINETTI NATALIA; IACONO RUBEN F; URETA DANIELA B; VALDEZ SILVINA N
Revista:
ANALYST
Editorial:
ROYAL SOC CHEMISTRY
Referencias:
Lugar: Cambridge; Año: 2014 vol. 139 p. 3017 - 3025
ISSN:
0003-2654
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 the 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
streptavidin-dichlorotriazinyl 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 those of 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 represents a cost-effective
alternative to traditional GADA determination by RBA and/or ELISA,
making it applicable to most medium-complexity laboratories