RESUMO
Introduction: The combined presence of autoantibodies to the 65 kDa isoform of glutamic acid decarboxylase (GADA) and to the islet-specific cation efflux transporter ZnT8 (ZnT8A) in serum is the best predictive sign of the loss of immune tolerance and the clinical manifestation of autoimmune diabetes mellitus (DM). The screening of GADA and ZnT8A could help to reach to a correct diagnosis and to start an early and adequate treatment. The aim of the study was to develop an immunoassay for the simultaneous detection of these autoantibodies using a chimera molecule that includes the immunodominant regions of ZnT8 and GAD65, expressed by baculovirus-insect cells system. Materials and Methods: ZnT8/GAD65 was expressed using the Bac to Bac™ baculovirus expression system. The recombinant chimera was purified by an His6-tag and identified by SDS-PAGE and western blot analysis, and by an indirect ELISA using specific antibodies against ZnT8 and GAD65. A fraction of ZnT8/GAD65 was biotinylated. A bridge ELISA (b-ELISA) was developed using ZnT8/GAD65 immobilized in polystyrene microplates, human sera samples from healthy individuals (n = 51) and diabetic patients (n = 49) were then incubated, and afterwards ZnT8/GAD65-biotin was added. Immune complexes were revealed with Streptavidin-Horseradish Peroxidase. Results were calculated as specific absorbance and expressed as standard deviation scores: SDs. Results: ZnT8/GAD65 was efficiently produced, yielding 30 mg/L culture medium, 80% pure. This recombinant chimera retains the immunoreactive conformation of the epitopes that are recognized by their specific antibodies, so it was used for the development of a high sensitivity (75.51%) and specificity (98.04%) b-ELISA for the detection of ZnT8A and/or GADA, in a one-step screening assay. The ROC curves demonstrated that this method had high accuracy to distinguish between samples from healthy individuals and diabetic patients (AUC = 0.9488); the cut-off value was stablished at 2 SDs. Conclusions: This immunoassay is useful either to confirm autoimmune diabetes or for detection in routine screening of individuals at risk of autoimmune DM. As DM is a slow progress disease, remaining asymptomatic for a long preclinical period, serological testing is of importance to establish a preventive treatment.
Assuntos
Diabetes Mellitus Tipo 1 , Humanos , Diabetes Mellitus Tipo 1/diagnóstico , Glutamato Descarboxilase , Imunoensaio , Complexo Antígeno-Anticorpo , AutoanticorposRESUMO
La estandarización de la hemoglobina A1c (HbA1c) permitió en algunos países su uso para el diagnóstico de la diabetes mellitus (DM) y la prediabetes, además de su empleo en el seguimiento del paciente con DM. Es importante recordar que se trata de una medida indirecta del promedio glucémico durante el tiempo de vida media del eritrocito, pudiendo verse afectada por variables no glucémicas, como también por interferencias analíticas según la metodología empleada para su determinación. A continuación, se describen las recomendaciones y consideraciones a tener en cuenta para la determinación de la HbA1c cuando se emplea como criterio diagnóstico de la DM, teniendo en cuenta que al utilizarla para tal fin es necesario que la medida se realice con métodos certificados y estandarizados al ensayo utilizado en el Diabetes Control and Complications Trial (DCCT).
The standardization of hemoglobin A1c (HbA1c) allowed in some countries its use for the diagnosis of diabetes mellitus (DM) and prediabetes, in addition to its use in the follow-up of patients with DM. It is important to highlight that it is an indirect measurement of the glycemic average during the halflife of the erythrocyte, and may be affected by non-glycemic variables, as well as by analytical interferences depending on the methodology used for its determination. The recommendations and considerations to take into account for the determination of HbA1c when it is used as a diagnostic criterion for diabetes are described below. In addition, it is important to emphasize that it is essential that the HbA1c measurement be performed with certified and standardized methods to the Diabetes Control and Complications Trial (DCCT)results.
Assuntos
Estado Pré-Diabético , Diabetes MellitusRESUMO
Establecer el punto de corte entre la glucemia en ayunas normal y la alterada resulta de suma importancia a los efectos de considerar a un paciente en riesgo, tanto de progresar a estdos más avanzados de la enfermedad como de sufrir complicaciones micro y macroangiopáticas. Desde 2006 la Sociedad Argentina de Diabetes (SAD), sobre la base de la evidencia considerada en ese momento, estableció el límite inferior de la glucemia alterada en ayunas (GAA) en 110 mg/dl; posteriormente, durante 2022, la Comisión Directiva de la SAD convocó a un grupo de expertos con el objeto de evaluar si esta recomendación debía mantenerse o, al igual que otras sociedades científicas de prestigio, adoptar a tal efecto 100 mg/dl. En este documento de Opiniones y Recomendaciones se encuentran los fundamentos por los cuales la SAD adoptará, de ahora en más, 100 mg/dl como límite inferior de la GAA, en base a las nuevas evidencias científicas que muestran que desde este punto de corte se produce un aumento en la progresión a la diabetes mellitus y de las complicaciones tanto macro como microangiopáticas.
To establish the cut-off point between normal and impaired fasting glycemia (IFG) is extremely important for the purposes of considering a patient at risk both of progressing to more advanced stages of the disease and of suffering micro- and macroangiopathic complications. Since 2006, the Argentine Diabetes Society (ADS), based on the evidence considered at that time, established the lower limit of IFG at 110 mg/d, laterduring the year 2022, The Board of Directors of the ADS vened a group of experts in order to assess whether this recommendation should be maintained or, like other prestigious scientific societies, adopt 100 mg/dl for this purpose. This Opinions and Recommendations document contains therationale for which the SAD will adopt, from now on, 100 mg/dlas the lower limit of the IFG, based on the new scientific edence that shows that from this cut-off point it produces increase in progression to diabetes and both macro and microangiopathic complications.
Assuntos
Estado Pré-DiabéticoRESUMO
Insect cell-baculovirus expression vector system is one of the most established platforms to produce biological products, and it plays a fundamental role in the context of COVID-19 emergency, providing recombinant proteins for treatment, diagnosis, and prevention. SARS-CoV-2 infection is mediated by the interaction of the spike glycoprotein trimer via its receptor-binding domain (RBD) with the host's cellular receptor. As RBD is required for many applications, in the context of pandemic it is important to meet the challenge of producing a high amount of recombinant RBD (rRBD). For this reason, in the present study, we developed a process based on Sf9 insect cells to improve rRBD yield. rRBD was recovered from the supernatant of infected cells and easily purified by metal ion affinity chromatography, with a yield of 82% and purity higher than 95%. Expressed under a novel chimeric promoter (polh-pSeL), the yield of rRBD after purification was 21.1 ± 3.7 mg/L, which is the highest performance described in Sf9 cell lines. Finally, rRBD was successfully used in an assay to detect specific antibodies in COVID-19 serum samples. The efficient strategy herein described has the potential to produce high-quality rRBD in Sf9 cell line for diagnostic purpose.
Assuntos
COVID-19 , SARS-CoV-2 , Animais , SARS-CoV-2/genética , Baculoviridae/genética , Ligação Proteica , Insetos , Glicoproteína da Espícula de CoronavírusRESUMO
La diabetes mellitus (DM) es una enfermedad heterogénea que presenta fenotipos clínicos diversos, todos con hiperglucemia. Históricamente se han utilizado cuatro factores para identificar esta diversidad: la edad de inicio, la gravedad de la enfermedad o grado de pérdida de la función de la célula beta, el grado de resistencia a la insulina y la presencia de autoanticuerpos asociados a la enfermedad. Actualmente, los parámetros empleados para clasificar los diferentes tipos de DM dificultan el diagnóstico y tratamiento de los pacientes. Las distintas presentaciones clínicas requieren una clasificación diagnóstica más eficaz para un abordaje terapéutico más preciso, valiéndose del avance de la inmunogenética y la bioquímica clínica. Esta guía está orientada a clasificar con precisión las presentaciones clínicas que a menudo generan incertidumbre dentro de los dos tipos principales de DM.
Diabetes mellitus (DM) is a heterogeneous disease, with diverse clinical phenotypes, all with hyperglycemia. Historically, four factors have been used to identify this diversity: the age at onset, the severity of the disease, that is, the degree of loss of beta cell function and insulin resistance, and the presence of circulating autoantibodies. Currently, the parameters used to classify the different types of DM make it difficult to diagnose and treat patients. The different clinical manifestations require an accurate diagnosis to achieve an effective therapeutic approach through the use of immunogenetics and clinical biochemistry. This practical guide aims to accurately classify the often uncertain clinical presentations within the two main types of diabetes.
Assuntos
Diabetes Mellitus , Autoanticorpos , Autoimunidade , GenéticaRESUMO
La diabetes mellitus (DM) es una enfermedad heterogénea que presenta fenotipos clínicos diversos, todos con hiperglucemia. Históricamente se han utilizado cuatro factores para identificar esta diversidad: la edad de inicio, la gravedad de la enfermedad o grado de pérdida de la función de la célula beta, el grado de resistencia a la insulina y la presencia de autoanticuerpos asociados a la enfermedad. Actualmente, los parámetros empleados para clasificar los diferentes tipos de DM dificultan el diagnóstico y tratamiento de los pacientes. Las distintas presentaciones clínicas requieren una clasificación diagnóstica más eficaz para un abordaje terapéutico más preciso, valiéndose del avance de la inmunogenética y la bioquímica clínica. Esta guía está orientada a clasificar con precisión las presentaciones clínicas que a menudo generan incertidumbre dentro de los dos tipos principales de DM.
Diabetes mellitus (DM) is a heterogeneous disease, with diverse clinical phenotypes, all with hyperglycemia. Historically, four factors have been used to identify this diversity: the age at onset, the severity of the disease, that is, the degree of loss of beta cell function and insulin resistance, and the presence of circulating autoantibodies. Currently, the parameters used to classify the different types of DM make it difficult to diagnose and treat patients. The different clinical manifestations require an accurate diagnosis to achieve an effective therapeutic approach through the use of immunogenetics and clinical biochemistry. This practical guide aims to accurately classify the often uncertain clinical presentations within the two main types of diabetes.
Assuntos
GenéticaRESUMO
Serology testing for COVID-19 is important in evaluating active immune response against SARS-CoV-2, studying the antibody kinetics, and monitoring reinfections with genetic variants and new virus strains, in particular, the duration of antibodies in virus-exposed individuals and vaccine-mediated immunity. In this study, recombinant S protein of SARS-CoV-2 was expressed in Rachiplusia nu, an important agronomic plague. One gram of insect larvae produces an amount of S protein sufficient for 150 determinations in the ELISA method herein developed. We established a rapid production process for SARS-CoV-2 S protein that showed immunoreactivity for anti-SARS-CoV-2 antibodies and was used as a single antigen for developing the ELISA method with high sensitivity (96.2%) and specificity (98.8%). Our findings provide an efficient and cost-effective platform for large-scale S protein production, and the scale-up is linear, thus avoiding the use of complex equipment like bioreactors.
Assuntos
Teste Sorológico para COVID-19 , COVID-19/diagnóstico , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/biossíntese , Animais , Larva/metabolismo , Larva/virologia , Nucleopoliedrovírus , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , SARS-CoV-2/metabolismo , Células Sf9 , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , SpodopteraRESUMO
Introduction: Insulin and proinsulin autoantibodies (IAA/PAA) are usually the first markers to appear in patients with type 1 Diabetes Mellitus (T1DM) and their prevalence ranges from 10 to 60% in the child-adolescent population. The reference method for IAA/PAA detection is the Radioligand Binding Assay (RBA), a highly specific and sensitive technique, but expensive and polluting. The aim of this work was to develop a novel flow cytometric microsphere-based immunoassay (FloCMIA) for PAA detection, employing recombinant human proinsulin (PI), as an alternative method to RBA, less expensive and harmful to the environment. Materials and Methods: Human PI was expressed as Thioredoxin fusion protein (TrxPI) in E. coli and a fraction was biotinylated. A double paratope model was used in which samples were incubated with TrxPI-biotin and microspheres adsorbed with TrxPI. The immune complexes were revealed using Streptavidin-Phycoerythrin. The geometric mean of the signals was analyzed, and the results were expressed as Standard Deviation scores (SDs). Sera from 100 normal human control and from 111 type 1 diabetic patients were evaluated by FloCMIA. To correlate the novel assay with RBA, 51 diabetic patients were selected, spanning a wide range of PAA reactivity by RBA. Results: The study of ROC curves allowed choosing a cut-off value of 3.0 SDs and the AUC was 0.705, indicating that FloCMIA has fair ability to distinguish between samples from each group. A prevalence of 50% for PAA was obtained in the population of diabetic patients studied. The specificity was 96% and the analytical sensitivity (percentage of patients RBA positive, also positive by FloCMIA) was 69%. There was a substantial agreement between methods (kappa statistic=0.700). Conclusions: A novel immunoassay based on flow cytometry that uses easy-to produce recombinant PI was developed. This assay constitutes an innovative and cost-effective alternative to RBA for the determination of PAA in patients' sera. The method developed here, presents good performance and a wide dynamic range together with a small required sample volume. Furthermore, these results make it possible to develop multiplex immunoassays that allow the combined detection of autoantibodies present in T1DM and other related autoimmune diseases.
Assuntos
Autoanticorpos/imunologia , Autoantígenos/metabolismo , Diabetes Mellitus Tipo 1/imunologia , Escherichia coli/metabolismo , Citometria de Fluxo/métodos , Proinsulina/imunologia , Proinsulina/metabolismo , Adolescente , Adulto , Autoanticorpos/sangue , Autoantígenos/genética , Biomarcadores/sangue , Estudos de Casos e Controles , Criança , Pré-Escolar , Diabetes Mellitus Tipo 1/sangue , Escherichia coli/genética , Feminino , Humanos , Imunoensaio/métodos , Lactente , Masculino , Microesferas , Pessoa de Meia-Idade , Proinsulina/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Adulto JovemRESUMO
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.
Assuntos
Autoanticorpos/sangue , Diabetes Autoimune Latente em Adultos/imunologia , Diabetes Autoimune Latente em Adultos/patologia , Idade de Início , Idoso , Idoso de 80 Anos ou mais , Autoanticorpos/análise , Autoimunidade/fisiologia , Biomarcadores , Estudos Transversais , Feminino , Glutamato Descarboxilase/imunologia , Humanos , Diabetes Autoimune Latente em Adultos/diagnóstico , Diabetes Autoimune Latente em Adultos/epidemiologia , Masculino , Monoéster Fosfórico Hidrolases/imunologia , Prognóstico , Transportador 8 de Zinco/imunologiaRESUMO
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.