Enzymatic Analysis, Quantitative Determination of L-Glutamic Acid by L-Glutamic Acid Decarboxylase (from E. coli)

Abstract Background: Autoantibodies for the 65-kDa form of glutamic acid decarboxylase (GAD65) and protein tyrosine phosphatase-like protein (IA-2) are measured for risk prediction and diagnosis of autoimmune diabetes mellitus. There is a lack of adequate nonisotopic alternatives to the most widely used method for both autoantibodies, which is a radiobinding assay (RBA). Methods: We compared two commercially available immunoassays, an ELISA and a time-resolved immunofluorometric assay (TR-IFMA), with RBA. Results: We found excellent agreement between the RBA and ELISA for measurement of GAD65 autoantibodies (GADAs); they showed comparable analytical precision in the cutoff range and achieved similar diagnostic specificity. The ELISA identified more GADA-positive individuals among patients with new-onset type 1 diabetes than did the RBA [89% (95% confidence interval, 78–95%), vs 71% (58–82%); P <0.03]. For IA-2 autoantibodies (IA-2As), only the TR-IFMA achieved analytical performance and diagnostic accuracy comparable to that of the RBA. These results with the GADA ELISA and IA-2A TR-IFMA were consistent with those obtained blindly in the Diabetes Antibody Standardization Program 2003. The performance of the GADA TR-IFMA and IA-2A ELISA was unsatisfactory, and these tests were not subjected to clinical evaluation. Conclusions: The GADA ELISA and IA-2A TR-IFMA behave comparably with RBA and are thus suitable for use in the clinical laboratory.

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Determination of glutamic acid decarboxylase activity in rat brain

Analytical Biochemistry ◽

10.1016/0003-2697(68)90053-5 ◽

1968 ◽

Vol 24 (1) ◽

pp. 1-8 ◽

Cited By ~ 6

Author(s):

P.J. Lupien ◽

C.M. Hinse ◽

L. Berlinguet

Keyword(s):

Glutamic Acid ◽

Rat Brain ◽

Glutamic Acid Decarboxylase ◽

Acid Decarboxylase ◽

Decarboxylase Activity

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Fluorimetric Assay for the Determination of Glutamic Acid Decarboxylase Activity in Subregions of Rat Brain Tissue

Analytical Letters ◽

10.1080/00032718008081370 ◽

1980 ◽

Vol 13 (15) ◽

pp. 1333-1344 ◽

Cited By ~ 6

Author(s):

Mack R. Holdiness ◽

Joseph B. Justice ◽

Darryl B. Neill ◽

John D. Salamone

Keyword(s):

Glutamic Acid ◽

Rat Brain ◽

Brain Tissue ◽

Glutamic Acid Decarboxylase ◽

Acid Decarboxylase ◽

Decarboxylase Activity ◽

Fluorimetric Assay ◽

Rat Brain Tissue

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No Specific Reactivity to E. Coli Glutamic Acid Decarboxylase from Sera of Newly-Diagnosed Insulin Dependent Diabetic Patients

International Journal of Immunopathology and Pharmacology ◽

10.1177/039463200301600206 ◽

2003 ◽

Vol 16 (2) ◽

pp. 129-138

Author(s):

C. Konidaris ◽

P.G. Mitlianga ◽

G.K. Papadopoulos

Keyword(s):

Monoclonal Antibody ◽

Glutamic Acid ◽

Glutamic Acid Decarboxylase ◽

Solid Phase ◽

Normal Subjects ◽

Cross Reactivity ◽

Acid Decarboxylase ◽

Diabetic Patients ◽

Dependent Manner ◽

E Coli

The 65 kD isoform of Glutamic Acid Decarboxylase (GAD), is one of the major autoantigens in human type 1 diabetes mellitus. This enzyme shares aminoacid identity, in select regions already determined as antigenic with its counterpart from E. coli. We tested the reactivity of diabetic and normal sera and an E. coli GAD-specific monoclonal antibody (2D9) to E. coli GAD by solid phase and competition ELISA, as well as immunoblotting to check for cross-reactivity of autoantibodies to the two antigens. Specific antibodies for E. coli GAD are present in diabetics and normal subjects without any differences in frequency and titer. The reactivity of such antibodies in ELISA could be blocked in a dose-dependent manner by the addition of excess antigen in the liquid phase. Furthermore, the monoclonal antibody against E. coli GAD does not recognise human recombinant GAD65 in an ELISA. We conclude that there is no basis for cross-reactivity between the two antigens, and antibody reactivity to GAD65 in man cannot arise from cross-reactivity to the E. coli enzyme.

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