RESUMEN
Background: There is limited information about diabetes and thyroid related autoantibodies in children with type 1 diabetes (T1D) or their siblings in Sri Lanka. Objectives: To assess in T1D children and their unaffected siblings the prevalence of autoantibodies to (1) glutamic acid decarboxylase (GADA), insulinoma associated antigen-2 (IA-2A) and zinc transporter 8 (ZnT8A) using 3 Screen ICA™ (3-Screen) and individual ELISA assays; (2) insulin (IAA); and (3) thyroid peroxidase (TPOA), thyroglobulin (TgA) and the TSH receptor (TSHRA). Methods: We selected - (a) consecutive T1D children, and (b) their unaffected siblings of both sexes, from the T1D Registry at Lady Ridgeway Hospital, Colombo. Results: The median age (IQR) of 235 T1D children and 252 unaffected siblings was 11 (8.4, 13.2) and 9 (5.4, 14.9) years respectively, and the duration of T1D was 23 (7, 54) months. (1) T1D children (a) 79.1% were 3-Screen positive; (b) all 3-Screen positives were individual antibody positive (GADA in 74%; IA-2A 31.1%; ZnT8A 38.7%); (c) and were younger (p=0.01 vs 3-Screen negatives); (d) multiple autoantibodies were present in 45.1%; (e) IA-2A (p=0.002) and ZnT8A (p=0.006) prevalence decreased with T1D duration. (f) TPOA and TgA prevalence was higher in T1D children compared to unaffected siblings (28%, p=0.001 and 31%, p=0.004, respectively). (2) Unaffected siblings (a) 6.3% were 3-Screen positive (p=0.001 vs T1D), and 2.4% were positive for IAA; (b) four subjects had two diabetes related autoantibodies, one of whom developed dysglycaemia during follow-up. Conclusions: The 3-Screen assay, used for the first time in Sri Lankan T1D children and their siblings as a screening tool, shows a high prevalence of T1D related Abs with a high correlation with individual assays, and is also a helpful tool in screening unaffected siblings for future T1D risk. The higher prevalence of thyroid autoantibodies in T1D children is consistent with polyglandular autoimmunity.
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Diabetes Mellitus Tipo 1 , Masculino , Femenino , Humanos , Niño , Sri Lanka , Hermanos , Glándula Tiroides , Prevalencia , AutoanticuerposRESUMEN
AIM: The study aim was to evaluate the RSR 3 Screen ICA™ and 2 Screen ICA™ for detection of islet cell autoimmunity in healthy Swedish subjects and patients with newly diagnosed type 1 diabetes (T1D). METHODS: 3 Screen is designed for combined detection of autoantibodies to glutamic acid decarboxylase (GADA), to the islet antigen IA-2 (IA-2A) and to zinc transporter 8 (ZnT8A), while 2 Screen detects GADA and IA-2A. Serum samples from 100 T1D patients at onset and 200 healthy controls were studied. RESULTS: 3 Screen achieved 93% assay sensitivity and 97.5% specificity, while 2 Screen achieved 91% assay sensitivity and 98.5% specificity. Samples were also tested in assays for individual autoantibodies. There was only one 3 Screen positive healthy control sample (0.5%) that was positive for multiple autoantibodies (IA-2A and ZnT8A). In contrast, most of the 93 3 Screen positive patients were positive for multiple autoantibodies with 72% (67/93) positive for both GADA and IA-2A and 57% (53/93) positive for three autoantibodies (GADA, IA-2A and ZnT8A). Insulin autoantibodies (IAA, measured by radioimmunoassay) were positive in 13 patients and two healthy controls. CONCLUSION: 3 Screen achieved high sensitivity and specificity, suitable for islet cell autoimmunity screening in a healthy population. In the case of 3 Screen positivity, further assays for GADA, IA-2A and ZnT8A are required to check for multiple autoantibody positivity, a hallmark for progression to T1D. In addition, testing for IAA in children below two years of age is warranted.
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Proteínas de Transporte de Catión , Diabetes Mellitus Tipo 1 , Autoanticuerpos , Niño , Glutamato Descarboxilasa , Humanos , Suecia/epidemiologíaRESUMEN
BACKGROUND: Testing for beta cell autoantibodies is used for wide-scale identification of early stages of type 1 diabetes. This requires suitable screening assays. We aimed to establish screening that utilized a first step assay (3 Screen) able to detect autoantibodies to the target antigens glutamic acid decarboxylase-65 (GAD), insulinoma-associated antigen 2 (IA-2), and zinc transporter 8 (ZnT8) to identify children positive for multiple beta cell autoantibodies. METHODS: An ELISA format was used where plates were coated with a mixture of recombinant GAD, IA-2, and ZnT8325W/R-dimer molecules. The performance was determined in venous blood from 686 first-degree relatives of patients with type 1 diabetes, and 200 patients at onset of type 1 diabetes, and applied as a screening assay in capillary blood from 33,639 general population children. RESULTS: The 3 Screen assay sensitivity for detecting autoantibody-positive patients at onset of type 1 diabetes was similar to that achieved by separate radiobinding assays (RBAs) for antibodies to GAD, IA-2, and ZnT8. Results in venous and capillary serum were correlated (R = 0.987). At a threshold corresponding to the 98th centile (29.1 U/mL) of all 33,639 capillary samples, the 3 Screen was positive in 123 samples with two or more RBA-positive antibodies to insulin, GAD, IA-2, or ZnT8, 146 with one antibody, and 479 that were RBA negative for beta cell autoantibodies. CONCLUSION: A 3 Screen ELISA was developed that was suitable for first step screening of multiple beta cell autoantibodies in capillary blood.
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Autoanticuerpos/sangre , Capilares , Diabetes Mellitus Tipo 1/inmunología , Ensayo de Inmunoadsorción Enzimática/métodos , Células Secretoras de Insulina/inmunología , Adolescente , Proteínas de Transporte de Catión/inmunología , Niño , Femenino , Glutamato Descarboxilasa/inmunología , Humanos , Masculino , Proteínas Tirosina Fosfatasas Clase 8 Similares a Receptores/inmunología , Sensibilidad y Especificidad , Transportador 8 de ZincRESUMEN
3 Screen, a new ELISA for the combined measurement of autoantibodies to GAD65, to IA-2 and to ZnT8, has been developed and evaluated. In the assay serum samples were incubated (overnight; 2-8°C) in ELISA plate wells coated with GAD65, IA-2 and ZnT8 followed by a wash step and incubation with biotinylated GAD65, IA-2 and ZnT8 (1h; 2-8°C,). The assay was completed by addition of streptavidin-peroxidase and tetramethylbenzidine. Samples tested in the 3 Screen were also analysed in ELISAs and radiobinding assays for the three individual autoantibodies. 129/132 (98%) samples from newly diagnosed T1DM children and 1/100 non-diabetic children controls were positive in 3 Screen. There was good agreement between 3 Screen and the individual autoantibody assays. Dilution of positive samples showed good linearity characteristics. In the 2015 Islet Autoantibody Standardization Program 3 Screen achieved 94% sensitivity, 95.6% specificity and 0.948 area under curve by ROC analysis. 3 Screen provides a simple and sensitive method for combined measurement of three major diabetes associated autoantibodies in a single sample. The assay should be a useful tool for large scale population screening for individuals at risk of developing T1DM.
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Autoanticuerpos/sangre , Proteínas de Transporte de Catión/sangre , Diabetes Mellitus Tipo 1/sangre , Ensayo de Inmunoadsorción Enzimática/métodos , Glutamato Descarboxilasa/sangre , Islotes Pancreáticos/inmunología , Proteínas Tirosina Fosfatasas Clase 8 Similares a Receptores/sangre , Adolescente , Autoanticuerpos/inmunología , Proteínas de Transporte de Catión/inmunología , Niño , Preescolar , Diabetes Mellitus Tipo 1/diagnóstico , Diabetes Mellitus Tipo 1/inmunología , Ensayo de Inmunoadsorción Enzimática/normas , Femenino , Glutamato Descarboxilasa/inmunología , Glutamato Descarboxilasa/metabolismo , Humanos , Lactante , Masculino , Proteínas Tirosina Fosfatasas Clase 8 Similares a Receptores/inmunología , Proteínas Tirosina Fosfatasas Clase 8 Similares a Receptores/metabolismo , Transportador 8 de ZincRESUMEN
AIMS: A bridging-type ELISA for measuring autoantibodies to zinc transporter 8 (ZnT8A) was assessed using samples from different forms of diabetes mellitus. METHODS: ZnT8A were measured using an ELISA in patients with type 1 diabetes mellitus (T1DM; n=94), latent autoimmune diabetes of adulthood (LADA; n=51), type 2 diabetes mellitus (T2DM; n=59) and healthy blood donors (HBD; n=200). ZnT8A in ELISA and immunoprecipitation assays (IPA) using ZnT8 dimer (W325/R325) and monomers (W325, R325 and Q325) were compared. RESULTS: Inter- and intra-assay coefficients of variation (CV) were 7.1% and 1.7%, respectively (medium ZnT8A) and 8.5% and 2.7%, respectively (high ZnT8A). In the ELISA 51/94 (54.3%) T1DM, 16/51 (31.4%) LADA and 1/59 (1.7%) T2DM sera were ZnT8A positive. ROC analysis of T1DM and HBD for the ELISA showed 54% sensitivity and 99% specificity (cutoff 15u/mL) and AUC 0.80 (95% CI, 0.74-0.86). ELISA and IPA measurements were in very good agreement (r=0.856, k=0.889, n=204). Measurement of ZnT8A in addition to autoantibodies for GAD, IA-2 and insulin increased antibody positivity in T1DM by 4.3%, from 80.9% to 85.1%. CONCLUSIONS: The bridging-type ELISA is a convenient and reproducible method for determination of ZnT8A in serum. Measurement of ZnT8A increased autoantibody positivity in adult T1DM.
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Autoanticuerpos/sangre , Autoanticuerpos/inmunología , Proteínas de Transporte de Catión/inmunología , Diabetes Mellitus Tipo 1/sangre , Diabetes Mellitus Tipo 2/sangre , Ensayo de Inmunoadsorción Enzimática/métodos , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Estudios de Casos y Controles , Proteínas de Transporte de Catión/química , Diabetes Mellitus Tipo 1/inmunología , Diabetes Mellitus Tipo 2/inmunología , Femenino , Glutamato Descarboxilasa/inmunología , Humanos , Insulina/inmunología , Masculino , Persona de Mediana Edad , Multimerización de Proteína , Estructura Cuaternaria de Proteína , Adulto Joven , Transportador 8 de ZincRESUMEN
BACKGROUND: A sensitive ELISA for measurement of IA-2 autoantibodies has been developed and assessed. Also, a combination ELISA for detection of both GAD65 autoantibodies and IA-2 autoantibodies is described. METHODS: The IA-2 autoantibody assay is based on the ability of IA-2 autoantibodies to form a bridge between IA-2 intracellular fragment coated onto ELISA plate wells and liquid-phase IA-2 labelled with biotin. The combination ELISA uses plates coated with both IA-2 and GAD65 and a mixture of IA-2-biotin and GAD65-biotin. Assay sensitivity was assessed using the WHO reference (NIBSC 97/550) for islet cell antibodies. IA-2 autoantibody measurements by ELISA were compared with measurements in immunoprecipitation assays (IPAs) based on 125I or 35S labelled IA-2. Combination ELISA results were compared with results obtained for individual autoantibodies. RESULTS: As little as 15 units/mL of NIBSC 97/550 was detectable in the IA-2 autoantibody ELISA compared to 125 units/mL by 125I-IA-2 IPA. 110/216(51%) sera from patients with type 1 DM were positive in the IA-2 autoantibody ELISA while 97/216 (45%) and 91/216 (42%) were positive in the 125I-IA2 and 35S-IA-2 IPAs, respectively. The IA-2 autoantibody ELISA showed 100% specificity for type 1 DM. The combination ELISA was able to detect GAD65 and/or IA-2 autoantibodies in 183/216 (85%) diabetic sera and 183/216 (85%) were also found positive for autoantibodies to IA-2 and/or to GAD65 in the assays for individual antibodies. Autoantibody measurements in the individual autoantibody assays and in the combination ELISA showed good agreement by Pearson correlation (r=0.92, n=216, p<0.001) and by Bland and Altman analysis. CONCLUSIONS: Sensitive and specific ELISAs for measurement of autoantibodies to IA-2 and to a combination of IA-2 and GAD65 have been developed. These assays are suitable for screening large numbers of samples in diabetes prediction and prevention trials.
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Autoanticuerpos/sangre , Autoantígenos/inmunología , Ensayo de Inmunoadsorción Enzimática/métodos , Glutamato Descarboxilasa/inmunología , Isoenzimas/inmunología , Proteínas de la Membrana/inmunología , Proteínas Tirosina Fosfatasas/inmunología , Especificidad de Anticuerpos , Autoanticuerpos/inmunología , Autoantígenos/metabolismo , Biotina/química , Diabetes Mellitus/sangre , Glutamato Descarboxilasa/metabolismo , Humanos , Radioisótopos de Yodo , Isoenzimas/metabolismo , Proteínas de la Membrana/metabolismo , Proteína Tirosina Fosfatasa no Receptora Tipo 1 , Proteínas Tirosina Fosfatasas/metabolismo , Proteínas Tirosina Fosfatasas Clase 8 Similares a Receptores , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
The interaction between IgA tissue transglutaminase (tTG) antibodies (Abs) and 35S-labelled tTG produced in a transcription/translation (TnT) system with various amino acid (aa) deletions has been studied. These experiments showed that the tTG N-terminal aa 1-89 were important for tTG Ab binding in all 15 coeliac disease sera studied and the central residues (aa 401-491) were important for binding of tTG Abs in all but one sera. The contribution of C-terminal residues to tTG Ab binding varied in different coeliac sera but overall was less than the contributions of the N terminal and central regions. Mouse monoclonal antibodies (MAbs) to tTG were produced and the tTG aa sequences recognised by the MAbs determined using modified 35S-labelled tTG proteins. Analysis of the inhibiting effects of patient sera tTG Ab on binding of tTG MAbs to tTG confirmed the importance of the N-terminal and central regions of tTG in forming serum tTG Ab binding sites. Recombinant human tTG was expressed in yeast and purified to better than 95% homogeneity using MAb affinity chromatography as a final purification step. This material was highly suitable for use in an ELISA for tTGAb.
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Autoanticuerpos/inmunología , Enfermedades Autoinmunes/inmunología , Enfermedad Celíaca/inmunología , Epítopos/inmunología , Proteínas de Unión al GTP/inmunología , Transglutaminasas/inmunología , Adolescente , Adulto , Anciano , Anticuerpos Monoclonales/inmunología , Autoanticuerpos/sangre , Enfermedades Autoinmunes/diagnóstico , Enfermedad Celíaca/diagnóstico , Ensayo de Inmunoadsorción Enzimática , Femenino , Proteínas de Unión al GTP/genética , Humanos , Masculino , Persona de Mediana Edad , Proteína Glutamina Gamma Glutamiltransferasa 2 , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Transglutaminasas/genéticaRESUMEN
BACKGROUND: A new ELISA for 65 kDa isoform of glutamic acid decarboxylase autoantibodies (GAD(65) Abs), which depends on GAD(65) Ab acting divalently and forming a bridge between immobilized GAD(65) and liquid-phase GAD(65)-biotin, is described. METHODS: Sera (25 microl) were incubated in GAD(65)-coated ELISA plate wells followed by washing and incubation with GAD(65)-biotin. After a further wash step, GAD(65)-biotin bound was quantitated by addition of streptavidin peroxidase followed by tetramethylbenzidine. Assay calibration was with WHO reference preparation 97/550. RESULTS: Using a cut-off for positivity of 5 units/ml, sera from 0.7% (2/300) healthy blood donors (HBDs), 100% (39/39) selected type 1 diabetes mellitus (DM) patients, 1.6% (1/62) type 2 diabetes mellitus patients and 3% (4/119) autoimmune disease controls were GAD(65) Ab positive in the ELISA. Levels of positivity in an immunoprecipitation assay (IPA) based on 125I-labelled GAD(65) (cut-off 25 units/ml) were 1%, 82%, 0% and 3%, respectively. ELISA inter-assay coefficients of variation (n=12) were 7.3%, 3.8%, 7.2% and 6.3% at 5.4, 40.8, 137 and 382 units/ml, respectively. CONCLUSIONS: The ELISA we have described has sensitivity and specificity at least as high as current radioactive assays. It has good precision and handling making it suitable for routine use.