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 LL, TRABUCCHI A, FACCINETTI NI, IACONO RF, URETA DB, POSKUS E, VALDEZ SN

ANALYST

ROYAL SOC CHEMISTRY

Lugar: CAMBRIDGE; Año: 2014 vol. 139 p. 3017 - 3025

0003-2654

The first measurable sign of arising autoimmunity in type1 diabetes mellitus is the detection of autoantibodies against beta-cell antigens, such as glutamic aciddecarboxylase (GAD65). GAD65 autoantibodies (GADA) are usually measured by the Radioligand BindingAssay (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 Tecnol´ogicos de Alto Nivel(STAN-CONICET) were analyzed by RBA, ELISA and three alternative FloCMIA designs. Protocol C exhibitedthe highest specificity (97.8%) andsensitivity (97.4%) and a wide dynamic range (1.00?134.40 SDs). Samples obtained from 40 new-onset diabetic patients werealso analyzed to further evaluate the performance of protocol C. The latter protocol showed a sensitivity of 58.6% and a prevalence of 47.5%. Twopatients 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 andspecific, requiring a low sample volume; it is environmentally adequate, innovative and represents acost-effective alternative to traditional GADA determination by RBAand/or ELISA, making it applicable to most medium-complexity laboratories.