Isolation and Functional Characterization of Recombinant GAD65 Autoantibodies Derived by IgG Repertoire Cloning From Patients With Type 1 Diabetes
- 1Department of Internal Medicine, University of Ulm, Ulm, Germany
- 2Institute of Immunology and Allergology, Inselspital, Bern, Switzerland
- 3Department of Orthopedic Surgery, University of Heidelberg, Heidelberg, Germany
Abstract
The generation of human monoclonal autoantibodies is critical for understanding humoral immune responses in autoimmunity. In this study, we isolated the first human recombinant antibodies to glutamate decarboxylase (rGAD65ab) by IgG repertoire cloning, phage display of Fab fragments, and biopanning from two patients at onset of type 1 diabetes. We demonstrate that natural Ig heavy- and light-chain pairings of autoantibodies can be isolated by the recombinant approach and have a major selection advantage over other rGAD65ab. Among eight rGAD65ab, three (rGAD65ab A–C) displayed all functional and structural properties of known disease-related GAD65ab, including reactivity in the enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), islet cell antibody (ICA) test, and variable gene usage. Dominant epitope recognition was directed to the previously defined epitope EP-1 in the middle of GAD65, corroborating its immunodominance in the molecule. New features, such as assay-dependent GAD65 reactivity and new epitope recognition, were observed in two rGAD65ab (D and E). These antibodies were positive in the GAD65 ELISA and ICA test but not in the GAD65 RIA, providing the first examples for ICA with incongruent results in solid-phase and fluid-phase assays. In conclusion, phage display–derived antibodies reflected well the natural autoantibody response in type 1 diabetes and may allow further characterization of assay-dependent features of GAD65ab and the recognition of epitopes in solid- but not fluid-phase assays.
Footnotes
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Address correspondence and reprint requests to Wiltrud Richter, Department of Orthopedic Surgery, University of Heidelberg, Schlierbacher Landstrasse 200, 69118 Heidelberg, Germany. E-mail: wiltrud.richter{at}ok.uni-heidelberg.de
Received for publication 19 December 2000 and accepted in revised form 24 May 2001.
BSA, bovine serum albumin; ELISA, enzyme-linked immunosorbent assay; HRP, horseradish-peroxidase; IA2, islet antigen 2; ICA, islet cell antibody; MICA, monoclonal islet cell antibody; PBMC, peripheral blood mononuclear cells; PBS, phosphate-buffered saline; PBS-T, PBS–0.5% Tween PCR, polymerase chain reaction; Phab, recombinant phage; PMSF, phenylmethylsulfonyl fluoride; rFab, recombinant Fab fragments; rGAD65ab, recombinant antibody to GAD65; RIA, radioimmunoassay.
