{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,6,12]],"date-time":"2026-06-12T07:17:18Z","timestamp":1781248638026,"version":"3.54.1"},"reference-count":41,"publisher":"American Diabetes Association","issue":"9","content-domain":{"domain":["diabetesjournals.org"],"crossmark-restriction":true},"short-container-title":[],"published-print":{"date-parts":[[2001,9,1]]},"abstract":"<jats:p>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\u2013C) 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\u2013derived 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.<\/jats:p>","DOI":"10.2337\/diabetes.50.9.1976","type":"journal-article","created":{"date-parts":[[2007,3,6]],"date-time":"2007-03-06T19:04:22Z","timestamp":1173207862000},"page":"1976-1982","update-policy":"https:\/\/doi.org\/10.2337\/ada-journal-policies","source":"Crossref","is-referenced-by-count":8,"title":["Isolation and Functional Characterization of Recombinant GAD65 Autoantibodies Derived by IgG Repertoire Cloning From Patients With Type 1 Diabetes"],"prefix":"10.2337","volume":"50","author":[{"given":"Karin","family":"Jury","sequence":"first","affiliation":[{"name":"Department of Internal Medicine, University of Ulm, Ulm, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Petra","family":"So\u0308hnlein","sequence":"additional","affiliation":[{"name":"Department of Internal Medicine, University of Ulm, Ulm, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Monique","family":"Vogel","sequence":"additional","affiliation":[{"name":"Institute of Immunology and Allergology, Inselspital, Bern, Switzerland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Wiltrud","family":"Richter","sequence":"additional","affiliation":[{"name":"Department of Orthopedic Surgery, University of Heidelberg, Heidelberg, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1167","reference":[{"key":"2022031207053384000_R1","doi-asserted-by":"crossref","unstructured":"Baekkeskov S, Nielsen JH, Marner B, Bilde T, Ludvigsson J, Lernmark A: Autoantibodies in newly diagnosed diabetic children immunoprecipitate human pancreatic islet cell proteins. 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Nature\u2008347:151\u2013156, 1990 (published erratum appears in Nature 347:782, 1990)","DOI":"10.1038\/347151a0"},{"key":"2022031207053384000_R6","doi-asserted-by":"crossref","unstructured":"Rabin DU, Pleasic SM, Shapiro JA, Yoo-Warren H, Oles J, Hicks JM, Goldstein DE, Rae PM: Islet cell antigen 512 is a diabetes-specific islet autoantigen related to protein tyrosine phosphatases. J Immunol\u2008152:3183\u20133188, 1994","DOI":"10.4049\/jimmunol.152.6.3183"},{"key":"2022031207053384000_R7","doi-asserted-by":"crossref","unstructured":"Lan MS, Lu J, Goto Y, Notkins AL: Molecular cloning and identification of a receptor-type protein tyrosine phosphatase, IA-2, from human insulinoma. 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