RESUMEN
Introducción: la diabetes mellitus autoinmune (DMA) y la enfermedad celíaca (EC) son enfermedades crónicas, poligénicas y multifactoriales vinculadas con la disfunción del sistema inmune. Dado que es frecuente que un mismo paciente presente ambas patologías, la detección simultánea de los marcadores de autoinmunidad de DMA y EC sería una estrategia racional para mejorar el diagnóstico. Objetivos: desarrollar un inmunoensayo basado en citometría de flujo (FloCMIA multiplex) para la detección simultánea y discriminativa de marcadores de DMA (GADA e IA-2A) y de EC (tTgA). Materiales y métodos: las muestras analizadas consistieron en sueros provenientes de 35 individuos controles normales y 21 pacientes con diabetes mellitus tipo 1 (DM1). Se empleó un modelo de "doble paratope" incubando los sueros con una mezcla de microesferas de diferente fluorescencia interna, cada una adsorbida con un autoantígeno: TrxGAD, TrxIA-2 o H6-tTg, y una mezcla de dichos autoantígenos biotinilados. Los inmunocomplejos se detectaron con estreptavidina-ficoeritrina y se adquirió en un citómetro de flujo. Resultados: FloCMIA multiplex detectó GADA en el 76,2% de los pacientes e IA-2A en el 52,38% (sensibilidad analítica: 88,24 y 56,25% respectivamente,y especificidad: 85,71%) y tTgA en el 42,86% (sensibilidad analítica: 50,0%, y especificidad: 80,0%). Estos resultados se contrastaron con el ensayo de unión de radioligando para GADA e IA-2A y se detectaron 80,95 y 76,19% de los sueros respectivamente (especificidad: 100%), y con un ELISA para tTgA se detectó un 38,1% (especificidad: 97,1%). Conclusiones: FloCMIA multiplex permitió detectar y discriminar GADA, IA-2A y/o tTgA, -en un único acto analítico- en sueros de pacientes con DMA y/o EC. El novedoso inmunoensayo desarrollado simplifica el screening de la población a gran escala
Introduction: autoimmune diabetes mellitus (ADM) and celiac disease (CD) are chronic, polygenic and multifactorial diseases associated with immune system dysfunction. As it is frequent that a patient presents both pathologies, the simultaneous detection of autoimmunity markers of ADM and CD would be a rational strategy to improve the diagnosis. Objectives: to develop an immunoassay based on Flow Cytometry (FloCMIA multiplex) for the simultaneous and discriminative detection of markers for ADM (GADA and IA-2A) and CD (tTgA). Materials and methods: thirty five serum samples of control individuals and 21 type 1 diabetes mellitus (T1DM) patients were assayed. A "double bridge" model was used for the assay, incubating the serum samples with a mixture of microspheres containing different amount of internal fluorescence, each one adsorbed with an autoantigen: TrxGAD, TrxIA-2 or H6-tTg, and a mixture of the same biotinilated autoantigens. The immunocomplexes were detected using streptavidinphycoerytrin and then acquired in a flow cytometer. Results: FloCMIA multiplex detected GADA in 76.2% of the patients; IA-2A in 52.38% (analytical sensitivity: 88.24% and 56.25% respectively, and specificity: 85.71%) and tTgA in 42.86% (analytical sensitivity: 50.0%, and specificity: 80.0%). These results were compared with the radioligand binding assay for GADA and IA-2A, detecting 80.95% and 76.19% of the serum samples respectively (100% specificity), and with an ELISA for tTgA detecting 38.1% (97.1% specificity). Conclusions: FloCMIA multiplex allowed detecting and discriminating GADA, IA-2A and/or tTgA, -in a single assay- in serum samples of ADM and/or CD. The novel developed immunoassay simplifies the screening of the large scale population
Asunto(s)
Autoanticuerpos , Inmunoensayo , Enfermedad Celíaca , Diabetes MellitusRESUMEN
BACKGROUND: Latent autoimmune diabetes in adults (LADA) is determined by both a noninsulin-dependent clinical presentation and an autoimmune pathogenic process. Glutamic acid decarboxylase antibody (GADA) constitutes the most important marker, although IA-2A and ZnT8A also define LADA presentation. Type 2 diabetes mellitus (T2DM) is the most prevalent type particularly over 65 years old. Studies about autoimmunity in this age group are scarce. OBJECTIVE: The aim of this work was to determine whether three autoantibodies for diabetes autoimmunity were present in elderly T2DM patients, and to assess the distinctive clinical features of autoantibody-positive patients. RESEARCH DESIGN AND METHODS: We recruited 153 patients with diabetes with onset of diabetes after 65 years of age and a BMI under 30 kg/m2 . RESULTS: The prevalence of at least one of the autoantibodies was 15.68% (24/153). The most prevalent autoantibody was GADA with 8.49% (13/153), followed by ZnT8A with 6.50% (10/153) and IA2A with 1.96% (3/153). The autoimmunity-positive group presented higher HbA1c (7.01 ± 1.98 vs 6.35 ± 1.01; P = 0.007) and more prevalent insulin therapy (25% vs 10.85%; P = 0.047). GADA-positive patients with diabetes presented higher FPG (7.79 ± 3.79 mmol/L vs 6.43 ± 1.6 mmol/L; P = 0.014) and insulin therapy more frequently (46% vs 10.71%; p = 0.015). GADA titre levels in the individuals with BMI under 27 kg/m2 were higher (35.00 ± 4.20) than those in the group with BMI over 27 kg/m2 (8.83 ± 3.041; P = 0.0005). CONCLUSION: Autoantibodies GADA and Znt8A may be useful markers in identifying a subgroup of older patients with a clinical presentation of diabetes which could be characterized as latent autoimmune diabetes in the elderly.
Asunto(s)
Autoanticuerpos/sangre , Diabetes Autoinmune Latente del Adulto/inmunología , Diabetes Autoinmune Latente del Adulto/patología , Edad de Inicio , Anciano , Anciano de 80 o más Años , Autoanticuerpos/análisis , Autoinmunidad/fisiología , Biomarcadores , Estudios Transversales , Femenino , Glutamato Descarboxilasa/inmunología , Humanos , Diabetes Autoinmune Latente del Adulto/diagnóstico , Diabetes Autoinmune Latente del Adulto/epidemiología , Masculino , Monoéster Fosfórico Hidrolasas/inmunología , Pronóstico , Transportador 8 de Zinc/inmunologíaRESUMEN
Autoimmune Diabetes Mellitus (DM) is a chronic disease caused by the selective destruction of insulin producing beta cells in human pancreas. DM is characterized by the presence of autoantibodies that bind a variety of islet-cell antigens. The 65 kDa isoform of glutamate decarboxylase (GAD65) is a major autoantigen recognized by these autoantibodies. Autoantibodies to GAD65 (GADA) are considered predictive markers of the disease when tested in combination with other specific autoantibodies. In order to produce reliable immunochemical tests for large scale screening of autoimmune DM, large amounts of properly folded GAD65 are needed. Herein, we report the production of human GAD65 using the baculovirus expression system in two species of larvae, Rachiplusia nu and Spodoptera frugiperda. GAD65 was identified at the expected molecular weight, properly expressed with high yield and purity in both larvae species and presenting appropriate enzymatic activity. The immunochemical ability of recombinant GAD65 obtained from both larvae to compete with [35S]GAD65 was assessed qualitatively by incubating GADA-positive patients' sera in the presence of 1 µM of the recombinant enzyme. All sera tested became virtually negative after incubation with antigen excess. Besides, radiometric quantitative competition assays with GADA-positive patients' sera were performed by adding recombinant GAD65 (0.62 nM-1.4 µM). All dose response curves showed immunochemical identity between proteins. In addition, a bridge-ELISA for the detection of GADA was developed using S. frugiperda-GAD65. This assay proved to have 77.3% sensitivity and 98.2% of specificity. GAD65 could be expressed in insect larvae, being S. frugiperda the best choice due to its high yield and purity. The development of a cost effective immunoassay for the detection of GADA was also afforded.
Asunto(s)
Autoanticuerpos/inmunología , Autoantígenos/inmunología , Diabetes Mellitus Tipo 1/diagnóstico , Glutamato Descarboxilasa/genética , Glutamato Descarboxilasa/inmunología , Animales , Autoantígenos/biosíntesis , Autoantígenos/genética , Baculoviridae/genética , Diabetes Mellitus Tipo 1/inmunología , Glutamato Descarboxilasa/biosíntesis , Humanos , Inmunoensayo/métodos , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Spodoptera/genética , Spodoptera/metabolismoRESUMEN
BACKGROUND: In the present work we described the recombinant production and characterization of heterodimeric construction ZnT8-Arg-Trp325 fused to thioredoxin using a high-performance expression system such as Escherichia coli. In addition, we apply this novel recombinant antigen in a non-radiometric method, with high sensitivity, low operational complexity and lower costs. RESULTS: ZnT8 was expressed in E. coli as a fusion protein with thioredoxin (TrxZnT8). After 3 h for induction, recombinant protein was obtained from the intracellular soluble fraction and from inclusion bodies and purified by affinity chromatography. The expression and purification steps, analyzed by SDS-PAGE and western blot, revealed a band compatible with TrxZnT8 expected theoretical molecular weight (≈ 36.8 kDa). The immunochemical ability of TrxZnT8 to compete with [35S]ZnT8 (synthesized with rabbit reticulocyte lysate system) was assessed qualitatively by incubating ZnT8A positive patient sera in the presence of 0.2-0.3 µM TrxZnT8. Results were expressed as standard deviation scores (SDs). All sera became virtually negative under antigen excess (19.26-1.29 for TrxZnT8). Also, radiometric quantitative competition assays with ZnT8A positive patient sera were performed by adding TrxZnT8 (37.0 pM-2.2 µM), using [35S]ZnT8. All dose-response curves showed similar protein concentration that caused 50% inhibition (14.9-0.15 nM for TrxZnT8). On the other hand, preincubated bridge ELISA for ZnT8A detection was developed. This assay showed 51.7% of sensitivity and 97.1% of specificity. CONCLUSIONS: It was possible to obtain with high-yield purified heterodimeric construction of ZnT8 in E. coli and it was applied in cost-effective immunoassay for ZnT8A detection.
Asunto(s)
Autoanticuerpos/sangre , Diabetes Mellitus Tipo 1/inmunología , Escherichia coli/genética , Transportador 8 de Zinc/genética , Transportador 8 de Zinc/inmunología , Animales , Antígenos/genética , Antígenos/inmunología , Autoanticuerpos/inmunología , Ensayo de Inmunoadsorción Enzimática/economía , Ensayo de Inmunoadsorción Enzimática/métodos , Humanos , Conejos , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/aislamiento & purificación , Tiorredoxinas/química , Tiorredoxinas/genéticaRESUMEN
OBJECTIVE: In order to gain further knowledge of the structure of zinc transporter 8 (ZnT8) epitopes, we studied the role of the amino acid at position 325 in the antigen and its dimeric conformation for autoantibodies to ZnT8 (ZnT8A) recognition. METHODS: For this purpose, several ZnT8 C-terminal domain variants were designed: monomer carrying Arg325 or Trp325, homo-dimers ZnT8-Arg-Arg325 and ZnT8-Trp-Trp325, and hetero-dimer ZnT8-Arg-Trp325. Two groups of Argentinian diabetic patients were subjected to analysis using [(35)S]-ZnT8 variants by radioligand binding assay (RBA): i) 100 new-onset, insulin-dependent, type 1 diabetic patients and ii) 282 slowly progressing to insulin requirement, non-obese adult-onset diabetic patients. In addition, 50 type 1 diabetic patients and 100 normal control sera provided by the American Diabetes Association (ADA) were evaluated in order to calculate the sensitivity and specificity of ZnT8A assays for each antigenic variant. Other routine ß-cell autoantibodies were also tested by RBA. RESULTS: Of the 100 Argentinian type 1 diabetic patients, 65 were ZnT8A+. Out of them, 8 patients recognized all recombinant forms of ZnT8 and most patients (56) reacted against the heterodimer. Additionally, out of 282 non-obese adult-onset diabetic patients 46 were ZnT8A+, whereas 29 patients recognized only dimers. Besides, exclusive reactivity against ZnT8A was found in 9.0% for type 1 diabetes mellitus and 10.3% for non-obese adult-onset diabetic patients. CONCLUSIONS: Significantly higher signal values in RBA were obtained with the heterodimeric variant. An increased detection of humoral autoimmunity was found in both groups when ZnT8A was employed in combination with the other ß-cell autoantibodies. The inclusion of homodimeric immunoreactive peptides revealed the existence of quaternary structure-defined epitopes probably resembling the actual state of the autoantigen in vivo. Finally, the differential profiles of ZnT8A exhibited by type 1 and non-obese adult-onset diabetic patients suggest the different nature of autoimmune processes underlying both pathologies.
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Autoanticuerpos/sangre , Autoantígenos/química , Proteínas de Transporte de Catión/sangre , Diabetes Mellitus Tipo 1/inmunología , Diabetes Mellitus Tipo 2/inmunología , Radioinmunoensayo/métodos , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Argentina , Autoanticuerpos/química , Niño , Epítopos/química , Femenino , Humanos , Células Secretoras de Insulina/inmunología , Masculino , Persona de Mediana Edad , Conformación Proteica , Radioinmunoensayo/normas , Proteínas Recombinantes , Adulto Joven , Transportador 8 de ZincRESUMEN
The first measurable sign of arising autoimmunity in type 1 diabetes mellitus is the detection of autoantibodies against beta-cell antigens, such as glutamic acid decarboxylase (GAD65). GAD65 autoantibodies (GADA) are usually measured by the Radioligand Binding Assay (RBA). The aim of this work was to develop protocols of flow cytometric microsphere-based immunoassays (FloCMIA) which involved glutamic acid decarboxylase fused to thioredoxin (TrxGAD65) adsorbed on polystyrene microspheres. Detection of bound GADA was accomplished by the use of anti-human IgG-Alexa Fluor 488 (protocol A), anti-human IgG-biotin and streptavidin-dichlorotriazinyl aminofluorescein (DTAF) (protocol B) or TrxGAD65-biotin and streptavidin-DTAF (protocol C). Serum samples obtained from 46 patients assayed for routine autoantibodies at Servicios Tecnológicos de Alto Nivel (STAN-CONICET) were analyzed by RBA, ELISA and three alternative FloCMIA designs. Protocol C exhibited the highest specificity (97.8%) and sensitivity (97.4%) and a wide dynamic range (1.00-134.40 SDs). Samples obtained from 40 new-onset diabetic patients were also analyzed to further evaluate the performance of protocol C. The latter protocol showed a sensitivity of 58.6% and a prevalence of 47.5%. Two patients resulted positive only by FloCMIA protocol C and its SDs were higher than those of RBA and ELISA, showing a significantly wide dynamic range. In conclusion, FloCMIA proved to be highly sensitive and specific, requiring a low sample volume; it is environmentally adequate, innovative and represents a cost-effective alternative to traditional GADA determination by RBA and/or ELISA, making it applicable to most medium-complexity laboratories.
Asunto(s)
Diabetes Mellitus Tipo 1/inmunología , Citometría de Flujo/métodos , Glutamato Descarboxilasa/inmunología , Inmunoensayo/métodos , Microesferas , HumanosRESUMEN
In this study, the characterization of insulin (auto)antibodies has been described, mainly in terms of concentration (q), affinity (Ka) and Ig (sub)isotypes by Surface Plasmon Resonance (SPR) in two particular clinical cases of individuals with severe episodes of impaired glycemia. Subject 1 suffers from brittle diabetes associated with circulating insulin antibodies (IA) due to insulin treatment. Subject 2 has insulin autoantibodies (IAA) associated with hypoglycemia in spite of not being diabetic and not having ever received exogenous insulin therapy. After conventional screening for IA/IAA by radioligand binding assay (RBA), we further characterized IA/IAA in sera of both patients in terms of concentration (q), affinity (Ka) and Ig (sub)isotypes by means of SPR technology. In both cases, q values were higher and Ka values were lower than those obtained in type 1 diabetic patients, suggesting that IA/IAA:insulin immunocomplexes could be responsible for the uncontrolled glycemia. Moreover, subject 1 had a predominat IgG1 response and subject 2 had an IgG3 response. In conclusion, SPR technology is useful for the complete characterization of IA/IAA which can be used in special cases where the simple positive/negative determination is not enough to achieve a detailed description of the disease fisiopathology.
Asunto(s)
Diabetes Mellitus Tipo 1/sangre , Diabetes Mellitus Tipo 1/inmunología , Anticuerpos Insulínicos/sangre , Insulina/inmunología , Resonancia por Plasmón de Superficie , Adolescente , Anciano , Autoanticuerpos/sangre , Autoanticuerpos/inmunología , Niño , Femenino , Humanos , Lactante , MasculinoRESUMEN
Type 1 diabetes mellitus (DM) is characterized by autoimmune aggression against pancreatic beta cells resulting in absolute deficiency of insulin secretion. The first detectable sign of emerging autoimmunity during the preclinical asymptomatic period is the appearance of diabetes-related autoantibodies. In children at risk for type 1 DM, high-affinity Insulin autoantibodies reactive to proinsulin, are associated with diabetes risk. Autoantibodies are usually measured by radioligand binding assay (RBA) that provides quasi-quantitative values reflecting potency (product between concentration and affinity) of specific autoantibodies. Aiming to improve the characterization of the specific humoral immune response, we selected surface plasmon resonance (SPR) as an alternative method to measure proinsulin autoantibodies (PAA). This novel technology has allowed real time detection of antibodies interaction and kinetic analysis. Herein, we have employed SPR to characterize the PAA present in sera from 28 childhood-onset (mean age 8.31±4.20) and 23 adult-onset diabetic patients (≥65 years old, BMI<30) in terms of concentration and affinity. When evaluating comparatively samples from both groups, childhood-onset diabetic patients presented lower PAA concentrations and higher affinities (median 67.12×10(-9) M and 3.50×10(7) M(-1), respectively) than the adults (median 167.4×10(-9) M and 0.84×10(7) M(-1), respectively). These results are consistent with those from the reference method RBA (Standard Deviation score median 9.49 for childhood-onset group and 5.04 for adult-onset group) where the binding can be directly related to the intrinsic affinity of the antibody, suggesting that there is a different etiopathogenic pathway between both types of clinical presentation of the disease. This technology has shown to be a useful tool for the characterization of PAAs parameters as an alternative to radioimmunoassay, with high versatility and reproducibility associated to low occupational and environmental risk. However, this technology is not eligible for routine marker screening, but this is a powerful technique for a fine description of the thermodynamic parameters of antigen-antibody interaction.
Asunto(s)
Afinidad de Anticuerpos , Autoanticuerpos/sangre , Autoanticuerpos/inmunología , Diabetes Mellitus Tipo 1/sangre , Proinsulina/inmunología , Adulto , Anciano , Anciano de 80 o más Años , Niño , Preescolar , Diabetes Mellitus Tipo 1/inmunología , Femenino , Humanos , Masculino , Proinsulina/sangre , Resonancia por Plasmón de SuperficieRESUMEN
Autoantibodies to zinc transporter 8 (ZnT8A) constitute an additional marker of autoimmune diabetes, complementing those already used in diagnosis support. ZnT8A could also be found in latent autoimmune diabetes of adults (LADA). The aim of this study was to evaluate the prevalence of ZnT8A in adult-onset diabetic patients in Argentinian population. A total of 271 patients diagnosed for diabetes at mean age 53.4 ± 10.9, body mass index ≤ 30, without insulin treatment for the first year of disease, and initially classified as type 2 diabetic patients were tested for ZnT8A using cDNA plasmids encoding the C-terminal domains (aa 268-369) carrying 325Arg, 325Trp, and a dimeric cDNA construct carrying both 325Arg and 325Trp (ZnT8 Arg-Trp325). We also analyzed proinsulin autoantibodies (PAA), glutamic acid decarboxylase autoantibodies (GADA), and protein tyrosine phosphatase IA-2 autoantibodies (IA-2A). A subset of 101 patients was followed during 6 years in order to analyze insulin requirement. Out of the 271 patients, 22.1% presented at least one humoral marker, 2.6% were PAA+, 12.5% were GADA+, 3.3% were IA-2A+, and 10.7% were ZnT8A+. Among the latter, 7.0% were ZnT8A-Arg325, 51.7% were ZnT8A-Trp325, and 62.1% were ZnT8A-Arg-Trp325. Furthermore, the prevalence of autoantibodies in the group of patients treated with insulin (n = 18) was 55.6%. These results demonstrated that a significant proportion of autoimmune adult-onset diabetic patients presented ZnT8A as the only humoral marker. Between them, the higher prevalence was for ZnT8A-Trp325. We suggest that screening for LADA patients, best performed with a minimal set of marker determination, must include at least the screening of GADA and ZnT8A-Arg-Trp325.