Development of 2 alternative enzyme-linked immunosorbent assays for routine screening of glutamic acid decarboxylase autoantibodies

Villalba, Anabel; Valdez, Silvina; Iacono, Rubén Francisco; Poskus, Edgardo

doi:10.1016/j.cca.2006.07.017

Cited by 12 publications

(18 citation statements)

References 19 publications

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“…4. b). These results showed that ELISA had a sensitivity of 58.6%; and a prevalence of 42.5% (SDs from -1.18 to 12.25, median SDs = 0.99) ( Table 2), not near to those for RBA, but similar to data previously reported for this method 10,12 . When evaluated by FloCMIA protocol C, the prevalence of positive results for this population was 47.5% and the relative sensitivity obtained was 58.6% (median SDs = 2.375), a value that was lower than that for RBA.…”

Section: Integrated Resultssupporting

confidence: 89%

“…These results indicated that ELISA had a relative sensitivity of 69.6% for this sera collection (percentage of patients RBA positive that were ELISA positive) and 100% specificity when analysis of control human sera was performed (calculated as 100% minus the percentage of false positives). These data are similar to those reported previously 12 . The ELISA showed SDs ranging from 0.11 to 10.03, and a median SDs = 5.02 (Table 1).…”

Section: Discussionsupporting

confidence: 93%

“…In this sense, we have previously described the expression of human GAD65 in Escherichia coli as a soluble and properly folded fusion protein with thioredoxin (TrxGAD65) 14 . This method has allowed us to determine the presence of GADA by ELISA 12 , with a similar format to that of Palomer et al 15 . This assay depends on the ability of divalent autoantibodies to form a bridge between immobilized TrxGAD65 and biotinylated TrxGAD65 present in the fluid phase.…”

Section: Introductionmentioning

confidence: 99%

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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

Trabucchi

Faccinetti

et al. 2014

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Flow Cytometric microsphere-based immunoassays (FloCMIA), involving polystyrene microspheres adsorbed with glutamic acid decarboxylase (GAD65) fused to thioredoxin (TrxGAD65), are used to assess GAD65 autoantibodies (GADA) in diabetic patients' sera. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 Page 2 of 18 Analyst AbstractThe 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.

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Section: Integrated Resultssupporting

confidence: 89%

Section: Discussionsupporting

confidence: 93%

Section: Introductionmentioning

confidence: 99%

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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

Trabucchi

Faccinetti

et al. 2014

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“…Recientemente se ha diseñado un método ELISA para determinación conjunta de GAD e IA-2, parece ser que con buenos resultados. Obtienen un 85% de positivos con el ensayo combinado GAD-65/IA-2 25,26 .…”

Section: Discussionunclassified

Estudio de autoanticuerpos en el inicio de la diabetes autoinmune en nuestro medio mediante ELISA

Moreno

Rey

Ríos

et al. 2011

Revista del Laboratorio Clínico

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“…The protocol employed was based on those previously described [62–65]. White Polystyrene microplates (96 F Maxisorp white microwell, Thermoscientific, Rockford, IL, USA) were coated ON at 4 °C with 40.0 ng of purified TrxIA-2 ic per well, washed three times with PBS, blocked for 1.5 h with 200 μL of blocking solution, and washed five times.…”

Section: Methodsmentioning

confidence: 99%

Novel prokaryotic expression of thioredoxin-fused insulinoma associated protein tyrosine phosphatase 2 (IA-2), its characterization and immunodiagnostic application

et al. 2016

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BackgroundThe insulinoma associated protein tyrosine phosphatase 2 (IA-2) is one of the immunodominant autoantigens involved in the autoimmune attack to the beta-cell in Type 1 Diabetes Mellitus. In this work we have developed a complete and original process for the production and recovery of the properly folded intracellular domain of IA-2 fused to thioredoxin (TrxIA-2ic) in Escherichia coli GI698 and GI724 strains. We have also carried out the biochemical and immunochemical characterization of TrxIA-2icand design variants of non-radiometric immunoassays for the efficient detection of IA-2 autoantibodies (IA-2A).ResultsThe main findings can be summarized in the following statements: i) TrxIA-2ic expression after 3 h of induction on GI724 strain yielded ≈ 10 mg of highly pure TrxIA-2ic/L of culture medium by a single step purification by affinity chromatography, ii) the molecular weight of TrxIA-2ic (55,358 Da) could be estimated by SDS-PAGE, size exclusion chromatography and mass spectrometry, iii) TrxIA-2ic was properly identified by western blot and mass spectrometric analysis of proteolytic digestions (63.25 % total coverage), iv) excellent immunochemical behavior of properly folded full TrxIA-2ic was legitimized by inhibition or displacement of [35S]IA-2 binding from IA-2A present in Argentinian Type 1 Diabetic patients, v) great stability over time was found under proper storage conditions and vi) low cost and environmentally harmless ELISA methods for IA-2A assessment were developed, with colorimetric or chemiluminescent detection.Conclusions E. coli GI724 strain emerged as a handy source of recombinant IA-2ic, achieving high levels of expression as a thioredoxin fusion protein, adequately validated and applicable to the development of innovative and cost-effective immunoassays for IA-2A detection in most laboratories.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-016-0309-2) contains supplementary material, which is available to authorized users.

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Development of 2 alternative enzyme-linked immunosorbent assays for routine screening of glutamic acid decarboxylase autoantibodies

Cited by 12 publications

References 19 publications

Flow cytometric microsphere-based immunoassay as a novel non-radiometric method for the detection of glutamic acid decarboxylase autoantibodies in type 1 diabetes mellitus

Flow cytometric microsphere-based immunoassay as a novel non-radiometric method for the detection of glutamic acid decarboxylase autoantibodies in type 1 diabetes mellitus

Estudio de autoanticuerpos en el inicio de la diabetes autoinmune en nuestro medio mediante ELISA

Novel prokaryotic expression of thioredoxin-fused insulinoma associated protein tyrosine phosphatase 2 (IA-2), its characterization and immunodiagnostic application

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