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1.
Diabetes ; 73(4): 565-571, 2024 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-38232306

RESUMO

Autoantibodies to glutamate decarboxylase (GADA) are widely used in the prediction and classification of type 1 diabetes. GADA radiobinding assays (RBAs) using N-terminally truncated antigens offer improved specificity, but radioisotopes limit the high-throughput potential for population screening. Luciferase-based immunoprecipitation system (LIPS) assays are sensitive and specific alternatives to RBAs with the potential to improve risk stratification. The performance of assays using the Nanoluc luciferase (Nluc)-conjugated GAD65 constructs, Nluc-GAD65(96-585) and full length Nluc-GAD65(1-585), were evaluated in 434 well-characterized serum samples from patients with recent-onset type 1 diabetes and first-degree relatives. Nonradioactive, high-throughput LIPS assays are quicker and require less serum than RBAs. Of 171 relatives previously tested single autoantibody positive for autoantibodies to full-length GAD65 by RBA but had not progressed to diabetes, fewer retested positive by LIPS using either truncated (n = 72) or full-length (n = 111) antigen. The Nluc-GAD65(96-585) truncation demonstrated the highest specificity in LIPS assays overall, but in contrast to RBA, N-terminus truncations did not result in a significant increase in disease-specificity compared with the full-length antigen. This suggests that binding of nonspecific antibodies is affected by the conformational changes resulting from addition of the Nluc antigen. Nluc-GAD65(96-585) LIPS assays offer low-blood-volume, high-specificity GADA tests for screening and diagnostics.


Assuntos
Diabetes Mellitus Tipo 1 , Humanos , Glutamato Descarboxilase , Sensibilidade e Especificidade , Autoanticorpos , Luciferases/genética , Imunoprecipitação
2.
Acta Diabetol ; 55(3): 263-270, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29305766

RESUMO

AIMS: Insulin autoantibodies (IAA) are often the first marker of autoimmunity detected in children in the preclinical phase of type 1 diabetes (T1D). Currently, the vast majority of laboratories adopt the radiobinding micro-assay (RBA) for measuring IAA. Our aim was to replace RBA with a novel non-radioactive IAA Luciferase Immuno Precipitation System (LIPS) assay with improved performance. METHODS: We developed (pro)insulin antigens with alternative placements of a NanoLuc™ luciferase reporter (NLuc). Performance in LIPS was evaluated by testing sera from new onset T1D (n = 80), blood donors (n = 123), schoolchildren (n = 186), first-degree relatives (FDRs) from the Bart's Oxford family study (n = 53) and from the Belgian Diabetes Registry (n = 136), coded sera from the Islet Autoantibody Standardization Program (IASP) (T1D n = 50, blood donors n = 90). RESULTS: IAA LIPS based on B chain-NLuc proinsulin or B chain-NLuc insulin, in which NLuc was fused at the C-terminus of the insulin B chain, required only 2 µL of serum and a short incubation time, showed high concordance with RBA (Spearman r = 0.866 and 0.833, respectively), high assay performance (B chain-NLuc proinsulin ROC-AUC = 0.894 and B chain-NLuc insulin ROC-AUC = 0.916), and an adjusted sensitivity at 95% specificity ranking on par with the best assays submitted to the two most recent IASP workshops. In FDRs, the IAA LIPS showed improved discrimination of progressors to T1D compared to RBA. CONCLUSIONS: We established a novel high-performance non-radioactive IAA LIPS that might replace the current gold standard RBA and find wide application in the study of the IAA response in T1D.


Assuntos
Diabetes Mellitus Tipo 1/sangue , Diabetes Mellitus Tipo 1/diagnóstico , Corantes Fluorescentes/metabolismo , Imunoprecipitação/métodos , Anticorpos Anti-Insulina/análise , Luciferases/metabolismo , Autoanticorpos/sangue , Biomarcadores/análise , Biomarcadores/metabolismo , Estudos de Casos e Controles , Criança , Corantes Fluorescentes/análise , Células HEK293 , Humanos , Insulina/imunologia , Anticorpos Anti-Insulina/sangue , Valor Preditivo dos Testes , Prognóstico , Proinsulina/imunologia , Sensibilidade e Especificidade
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