Detection and immunochemical characterization of glutamic acid decarboxylase autoantibodies in patients with autoimmune diabetes mellitus

Abstract

Since GAD65 undergoes post-translational processing and targeting to subcellular compartments and membranes, it may exhibit different immunochemical properties in the cell context compared with the soluble protein expressed in the cell-free eukaryotic system used in the reference method for GADA assessment (radioligand binding assay (RBA)).

In the present work, we detected and characterized GADA in 72 sera from patients with type 1 diabetes mellitus (DM) and 14 sera from adult-onset diabetes patients using analytical systems in which GAD65 is expressed in a cellular context: confocal indirect immunofluorescence (IIF) and electron microscopy after immunogold labeling on monolayers of transfected Chinese hamster ovary (CHO) cells, and immunoprecipitation (IP) of metabolically labeled GAD65.

Eighteen serum samples, 16 from type 1 diabetes patients and two from adult-onset diabetes patients, were positive by confocal IIF but scored negative by RBA. All of these 18 sera immunoprecipitated a 65 kDa protein, supporting the existence of the GADA marker in such patients.

It may be concluded that GADA negativity by the conventional RBA method using the soluble antigen, as well as negativity for other common markers measured by similar methods, is not enough to rule out the existence of the specific autoimmune component in childhood or adult-onset diabetes. Other analytical methods based in a more physiological presentation of the autoantigen structure, as confocal IIF and IP, bring an extra support to assess the complete repertoire of specific autoantibodies to native-like and membrane-bound, or denatured, β-cell antigens.

• Diabetes mellitus
• glutamic acid decarboxylase
• immunofluorescence
• markers

Acknowledgements

We thank G. Krochik and C. Mazza at the Hospital Nacional de Pediatría J. P. Garrahan (Buenos Aires, Argentina) for collecting and providing type 1 diabetes patient sera. We are grateful to G. Frechtel and the Division of Hemoteraphy at the Hospital de Clínicas José de San Martín (Buenos Aires, Argentina) for providing sera from the adult-onset diabetes patients and control sera, respectively. We are grateful to Dr.Å. Lernmark, Karolinska Institutet, Dr A. Karlsen and Dr C. Grubin, University of Washington, Seattle, WA, USA, for providing pEx9 vectors carrying the glutamate decarboxylase gene used in RBA experiments. This work was supported in part by grants from National Research Council (CONICET, PIP 5214), and University of Buenos Aires (UBACYT B118), Buenos Aires, Argentina.