RESUMEN
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.
Asunto(s)
Autoanticuerpos/inmunología , Autoanticuerpos/aislamiento & purificación , Clonación Molecular , Diabetes Mellitus Tipo 1/inmunología , Glutamato Descarboxilasa/inmunología , Inmunoglobulina G/genética , Isoenzimas/inmunología , Adulto , Anticuerpos Monoclonales , Especificidad de Anticuerpos , Epítopos , Femenino , Genes de Inmunoglobulinas/fisiología , Humanos , Región Variable de Inmunoglobulina/genética , Biblioteca de Péptidos , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/aislamiento & purificaciónRESUMEN
AIMS/HYPOTHESIS: The aim of this study was to analyse the conformational and linear epitope profiles of glutamic acid decarboxylase antibody (GAD65-ab)-positive sera to find disease-specific epitope profiles and to study, whether GAD65-ab epitope recognition changes or spreads during the prediabetic period and, thus, can provide markers to differentiate early from later stages of progression to diabetes. METHODS: Sera from subjects before (n = 21), at onset (n = 44), or at increased risk of Type I (insulin-dependent) diabetes mellitus (n = 20) and from patients with stiff-man syndrome (SMS, n = 18) or polyendocrine autoimmune syndrome (PAS, n = 21) were analysed for conformational and linear GAD65 epitope recognition by an immunohistochemical blocking test based on human monoclonal GAD65-ab (MICA 1-10) and western blotting of a GAD65 epitope-cDNA-library. RESULTS: A redundant reactivity of many GAD65-ab positive sera to three major conformational (EP-1, EP-2, EP-3) and two dominant linear epitope clusters (amino acid 1-124 and 535-585) was observed in diabetes, polyendocrine autoimmune syndrome and stiff-man syndrome and no disease-specific epitopes or epitope-profiles were detected. Epitope recognition broadened with higher titres and with the vulnerability of patients to acquire additional autoimmune diseases apart from diabetes. Low GAD65-ab serum titres (< 1200 arbitrary units) and EP-1 recognition in the absence of EP-2 binding characterised the early immune response. Changing epitope profiles combined stable recognition of EP-1 with gain or loss of reactivity to C-terminal epitopes during follow-up. CONCLUSION/INTERPRETATION: A maturing autoantibody response, which could spread from EP-1-recognition to other regions of GAD65, resulted in titre-related rather than disease-specific epitope profiles which were not sufficient to predict whether GAD65-ab positive subjects will progress to Type I diabetes, autoimmune polyendocrine syndrome or stiff-man syndrome.
Asunto(s)
Diabetes Mellitus Tipo 1/inmunología , Glutamato Descarboxilasa/inmunología , Isoenzimas/inmunología , Estado Prediabético/inmunología , Adolescente , Adulto , Enfermedades Autoinmunes/sangre , Enfermedades Autoinmunes/inmunología , Niño , Preescolar , Diabetes Mellitus Tipo 1/sangre , Epítopos/inmunología , Femenino , Glutamato Descarboxilasa/química , Humanos , Lactante , Isoenzimas/química , Masculino , Persona de Mediana Edad , Estado Prediabético/sangre , Factores de Riesgo , Síndrome de la Persona Rígida/sangre , Síndrome de la Persona Rígida/inmunologíaRESUMEN
A purified, GPI-linked receptor complex isolated from Manduca sexta midgut epithelial cells was reconstituted in planar lipid bilayers. CryIAa, CryIAc and CryIC, three Bacillus thuringiensis insecticidal proteins, formed channels at much lower doses (0.33-1.7 nM) than in receptor-free membranes. The non-toxic protein CryIB also formed channels, but at doses exceeding 80 nM. The channels of CrylAc, the most potent toxin against M. sexta, rectified the passage of cations. All other toxin channels displayed linear current-voltage relationships. Therefore, reconstituted Cry receptors catalyzed channel formation in phospholipid membranes and, in two cases, were involved in altering their biophysical properties.