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1. Development of an immunoassay for the simultaneous detection of GADA and ZnT8A in autoimmune diabetes using a ZnT8/GAD65 chimeric molecule

Author

Aldana Trabucchi, Silvina Sonia Bombicino, Adriana Victoria Sabljic, Juan Ignacio Marfía, Alexandra Marisa Targovnik, Rubén Francisco Iacono, María Victoria Miranda, and Silvina Noemí Valdez

Subjects
autoimmune diabetes mellitus, ZnT8/GAD65 chimera, insect cells, autoantibodies, ELISA, Immunologic diseases. Allergy, RC581-607
Abstract

IntroductionThe 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 MethodsZnT8/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.ResultsZnT8/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.ConclusionsThis 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.

Published
2023
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2. Novel Flow Cytometric Immunoassay for Detection of Proinsulin Autoantibodies in Diabetes Mellitus Employing a Recombinant Autoantigen Expressed in E. coli

Author

Adriana Victoria Sabljic, Silvina Sonia Bombicino, Juan Ignacio Marfía, Luciano Lucas Guerra, Alberto Penas Steinhardt, Natalia Inés Faccinetti, Rubén Francisco Iacono, Edgardo Poskus, Aldana Trabucchi, and Silvina Noemí Valdez

Subjects
diabetes mellitus, autoimmunity, autoantibodies, proinsulin, flow cytometry, immunoassay, Immunologic diseases. Allergy, RC581-607
Abstract

IntroductionInsulin 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 MethodsHuman 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.ResultsThe 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).ConclusionsA 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.

Published
2021
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3. O2 Screening of anti-tissue transglutaminase autoantibodies in pediatric patients recently diagnosed with Diabetes Mellitus, employing a novel immunoassay

Author

Silvina Sonia Bombicino, Adriana Victoria Sabljic, Juan Ignacio Marfía, Liliana Trifone, Adriana Roussos, Aldana Trabucchi, Edgardo Poskus, and Silvina Valdez

Subjects
screening, diabetes, inmunoensayo., Nutritional diseases. Deficiency diseases, RC620-627, Diseases of the endocrine glands. Clinical endocrinology, RC648-665
Abstract

Introduction: Type 1 Diabetes Mellitus (DM1) and Celiac Disease (CD) are chronic, polygenic and multifactorial diseases associated to an immune system dysfunction. They are simultaneously presented in 10% of the patients, thus it is recommended the routine screening of CD in DM1 recently diagnosed patients. Objectives: Screening pediatric patients diagnosed with DM1 in order to evaluate the presence of anti-tissue transglutaminase antibodies (tTgA), using a method based in flow cytometry (FloCMIA: Flow Cytometric Microsphere based Immunoassay). Materials and Methods: The presence of tTgA in serum samples of 253 pediatric patients, recently diagnosed with DM1, was evaluated by FloCMIA. A "double paratope" model was used incubating the samples with polystyrene microspheres (5m) adsorbed with recombinant tissue transglutaminase (His6-tTg), ON at 4 ºC. The mixture was then incubated with biotinylated His6-tTg and the immunocomplexes formed were detected with streptavidin-Phycoerythrin and acquired in a Flow Cytometer. The results were expressed as Standard Deviation scores (SDs), considering as positive the samples with SDs > 1.7. Samples that were tTgA positive by FloCMIA were then contrasted using a commercial ELISA kit

Published
2020
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4. Rapid and cost-effective process based on insect larvae for scale-up production of SARS-COV-2 spike protein for serological COVID-19 testing

Author

Aldana Trabucchi, Matías Fingermann, Silvina Noemi Valdez, Osvaldo Cascone, María Gabriela López, Ruben F. Iacono, Susana Vázquez, Leonardo G. Alonso, Federico Javier Wolman, Silvina Sonia Bombicino, Joaquín Manuel Birenbaum, Juan Ignacio Marfía, Gregorio Juan Mc Callum, Juan Mauricio Minoia, María Victoria Miranda, Alexandra Marisa Targovnik, Oscar Taboga, Adriana Victoria Sabljic, and Ignacio Smith

Subjects
Insect Larvae, Testing, Bioengineering, insect larvae, Larvas de Insectos, Spodoptera, Applied Microbiology and Biotechnology, Virus, Article, law.invention, Serology, COVID-19 Serological Testing, Rachiplusia nu, Antigen, law, Immunity, Protein biosynthesis, Sf9 Cells, SPIKE, Animals, antibodies, purl.org/becyt/ford/3.4 [https], biology, SARS-CoV-2, SEROLOGY, fungi, Proteins, COVID-19, Articles, Severe Acute Respiratory Syndrome Coronavirus 2, biology.organism_classification, Virology, Nucleopolyhedroviruses, Recombinant Proteins, Ensayo, Larva, Proteínas, Spike Glycoprotein, Coronavirus, Recombinant DNA, biology.protein, SARS‐CoV‐2 coronavirus, purl.org/becyt/ford/3 [https], ELISA, trimeric spike, Antibody, Coronavirus del Síndrome Respiratorio Agudo Grave 2, Biotechnology
Abstract

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., A rapid and cost‐effective process for scale‐up the production of SARS‐CoV‐2 Spike protein was developed in this study. The authors obtained high‐quality protein (trimeric and glycosylated state) after one step of purification from Rachiplusia nu larvae. Our results demonstrate that the Spike protein produced in insect larvae is immunoreactive against the sera from COVID‐19 patients and can be used as a single antigen in immunoassays for the detection of anti‐SARS‐Cov‐2 antibodies

Published
2021

5. Expression of recombinant glutamic acid decarboxylase in insect larvae and its application in an immunoassay for the diagnosis of autoimmune diabetes mellitus

Author

Luciano Lucas Guerra, María Victoria Miranda, Adriana Victoria Sabljic, Silvina Sonia Bombicino, Silvina Noemi Valdez, Alexandra Marisa Targovnik, Natalia Ines Faccinetti, Juan Ignacio Marfía, Ruben F. Iacono, and Aldana Trabucchi

Subjects
0301 basic medicine, endocrine system, endocrine system diseases, medicine.medical_treatment, BACULOVIRUS, Glutamate decarboxylase, INSECT LARVAE, lcsh:Medicine, Biology, Spodoptera, Autoantigens, Article, law.invention, 03 medical and health sciences, 0302 clinical medicine, Antigen, law, AUTOINMUNE DIABETES MELLITUS, medicine, Animals, Humans, lcsh:Science, Autoantibodies, chemistry.chemical_classification, Immunoassay, Multidisciplinary, medicine.diagnostic_test, Glutamate Decarboxylase, Insulin, lcsh:R, fungi, Autoantibody, nutritional and metabolic diseases, purl.org/becyt/ford/3.1 [https], biology.organism_classification, Molecular biology, Recombinant Proteins, 030104 developmental biology, Enzyme, Diabetes Mellitus, Type 1, chemistry, Recombinant DNA, lcsh:Q, ELISA, purl.org/becyt/ford/3 [https], Baculoviridae, 030217 neurology & neurosurgery
Abstract

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. Fil: Trabucchi, Aldana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; Argentina Fil: Bombicino, Silvina Sonia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; Argentina Fil: Targovnik, Alexandra Marisa. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Nanobiotecnología. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Nanobiotecnología; Argentina Fil: Marfía, Juan Ignacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; Argentina Fil: Sabljic, Adriana Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; Argentina Fil: Faccinetti, Natalia Ines. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; Argentina Fil: Guerra, Luciano Lucas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; Argentina Fil: Iacono, Ruben Francisco. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; Argentina Fil: Miranda, Maria Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Nanobiotecnología. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Nanobiotecnología; Argentina Fil: Valdez, Silvina Noemi. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; Argentina

Published
2019

6. Heart mitochondrial dysfunction in diabetic rats

Author

Silvina Sonia Bombicino, Darío E. Iglesias, Laura Beatriz Valdez, I.A. Rukavina Mikusic, and Alberto Boveris

Subjects
0301 basic medicine, 03 medical and health sciences, 030104 developmental biology, business.industry, Medicine, General Medicine, business
Published
2016
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7. Superoxide and hydrogen peroxide productions by NO-inhibited complex III

Author

Laura B. Valdez, Darío E. Iglesias, Silvina Sonia Bombicino, and Alberto Boveris

Subjects
0301 basic medicine, Superoxide, General Medicine, Medicinal chemistry, law.invention, S-Nitrosoglutathione, 03 medical and health sciences, chemistry.chemical_compound, 030104 developmental biology, Spermine NONOate, chemistry, law, Coenzyme Q – cytochrome c reductase, Electron paramagnetic resonance, Hydrogen peroxide
Abstract

Fil: Iglesias, Dario E.. Universidad de Buenos Aires. Facultad de Farmacia y Bioquimica. Departamento de Quimica Analitica y Fisicoquimica; Argentina

Published
2016
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9. O2 Screening de autoanticuerpos anti-transglutaminasa tisular en pacientes pediátricos con diagnóstico reciente de diabetes mellitus, empleando un novedoso inmunoensayo

Author

Aldana Trabucchi, Liliana Trifone, Silvina Sonia Bombicino, Juan Ignacio Marfía, Silvina Noemi Valdez, Edgardo Poskus, Adriana Roussos, and Adriana Victoria Sabljic

Subjects
medicine.medical_specialty, Type 1 diabetes, Routine screening, medicine.diagnostic_test, biology, Tissue transglutaminase, business.industry, medicine.disease, Gastroenterology, Flow cytometry, Immune system, Internal medicine, Immunoassay, Diabetes mellitus, medicine, biology.protein, General Earth and Planetary Sciences, Antibody, business, General Environmental Science
Abstract

Introducción: la diabetes mellitus tipo 1 (DM1) y la enfermedad celíaca (EC) son enfermedades crónicas, poligénicas y multifactoriales vinculadas con una disfunción del sistema inmune. Se presentan en simultaneidad en un 10% de los pacientes, por lo cual se recomienda el screening de rutina para EC en pacientes con reciente diagnóstico de DM1.Objetivos: realizar un screening en pacientes pediátricos con diagnóstico de DM1 para evaluar la presencia de autoanticuerpos anti-transglutaminasa tisular (tTgA), utilizando un método basado en citometría de flujo (FloCMIA: Flow Cytometric Microsphere based Immunoassay).Materiales y métodos: se ensayaron sueros de 253 pacientes pediátricos con diagnóstico reciente de DM1 y se evaluó la presencia de tTgA mediante FloCMIA. Se empleó un modelo de doble paratope incubando los sueros con microesferas de poliestireno de 5 µm adsorbidas con la transglutaminasa tisular recombinante (His6tTg), ON a 4 ºC. La mezcla se incubó con His6tTg marcada con biotina y los inmunocomplejos formados se detectaron empleando estreptavidina-ficoeritrina y se adquirió en un citómetro de flujo. Los resultados fueron expresados en Standard Deviation scores (SDs), considerando positivas las muestras con SDs>1,7. Las muestras tTgA positivas por FloCMIA se contrastaron con un kit de ELISA comercial.

Published
2020
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10. INMUNOENSAYO MULTIPLEX PARA EL DIAGNÓSTICO SIMULTÁNEO DE DIABETES MELLITUS AUTOINMUNE Y ENFERMEDAD CELÍACA

Author

Juan Ignacio Marfía, Aldana Trabucchi, Adriana Victoria Sabljic, Ivana Masci, Silvina Sonia Bombicino, Silvina Noemi Valdez, Natalia Ines Faccinetti, Edgardo Poskus, and Luciano Lucas Guerra

Subjects
education.field_of_study, medicine.diagnostic_test, business.industry, Population, Enfermedad Celíaca, Autoanticuerpos, Disease, Inmunoensayo Multiplex, medicine.disease_cause, Diabetes Mellitus Autoinmune, Autoimmunity, Flow cytometry, purl.org/becyt/ford/3.3 [https], Immune system, Immunoassay, Autoimmune diabetes, Immunology, medicine, General Earth and Planetary Sciences, purl.org/becyt/ford/3 [https], Multiplex, education, business, General Environmental Science
Abstract

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. ntroduction: 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. Fil: Bombicino, Silvina Sonia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; Argentina Fil: Sabljic, Adriana Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; Argentina Fil: Faccinetti, Natalia Ines. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; Argentina Fil: Guerra, Luciano Lucas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; Argentina Fil: Marfía, Juan Ignacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; Argentina Fil: Masci, Ivana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; Argentina Fil: Trabucchi, Aldana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; Argentina Fil: Poskus, Edgardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; Argentina Fil: Valdez, Silvina Noemi. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; Argentina

Published
2020
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11. Mitochondrial peroxynitrite generation is mainly driven by superoxide steady-state concentration rather than by nitric oxide steady-state concentration

Author

Laura B. Valdez, Darío E. Iglesias, Silvina Sonia Bombicino, Alberto Boveris, and Ivana Rukavina Mikusic A

Subjects
0301 basic medicine, PEROXINITRITE, Bioenergetics, Radical, Mitochondrion, 010402 general chemistry, 01 natural sciences, Nitric oxide, Ciencias Biológicas, SUPEROXIDE, purl.org/becyt/ford/1 [https], 03 medical and health sciences, chemistry.chemical_compound, MITOCHONDRIA, purl.org/becyt/ford/1.6 [https], Autoxidation, Superoxide, STEADY-STATES, Bioquímica y Biología Molecular, Biofísica, 0104 chemical sciences, 030104 developmental biology, chemistry, Biophysics, Steady state (chemistry), Peroxynitrite, CIENCIAS NATURALES Y EXACTAS, NITRIC OXIDE
Abstract

In biological systems, ONOO- production depends on production rates of NO and O2-, and on the reactions of these two free radicals with other biological components, which limit the local concentrations of NO and O2-. In mitochondria, O2- is generated through the autoxidation of semiquinones at Complexes I and III, and it may suffer the SOD-catalysed dismutation reaction to produce H2O2 or react with NO in a classical termination reaction between free radicals. These diffusion-controlled reactions kinetically compete for O2- degradation. Results from our laboratory have shown that even in physiopathological situations in which NO production is reduced, such as the mitochondrial dysfunction associated to stunned heart, mitochondrial ONOO- production rate may be slightly increased if the steady-state concentration of O2- is augmented. The enhancement in O2- concentration leads to an increase in its degradation by reaction with NO, decreasing NO bioavailability and increasing ONOO- production rate. Therefore, mitochondrial ONOO- generation is mainly driven by O2- rather than by NO steady-state concentrations. In this scenario, the switch from signalling pathways of NO to oxidative damage takes place and oxidation reactions of biomolecules or modification of proteins by nitration may occur. Fil: Valdez, Laura Batriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Bioquímica y Medicina Molecular. Universidad de Buenos Aires. Facultad Medicina. Instituto de Bioquímica y Medicina Molecular; Argentina Fil: Bombicino, Silvina Sonia. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Iglesias, Dario Ezequiel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Bioquímica y Medicina Molecular. Universidad de Buenos Aires. Facultad Medicina. Instituto de Bioquímica y Medicina Molecular; Argentina Fil: Rukavina Mikusic, Ivana Agustina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Bioquímica y Medicina Molecular. Universidad de Buenos Aires. Facultad Medicina. Instituto de Bioquímica y Medicina Molecular; Argentina Fil: Boveris, Alberto Antonio. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Bioquímica y Medicina Molecular. Universidad de Buenos Aires. Facultad Medicina. Instituto de Bioquímica y Medicina Molecular; Argentina

Published
2018

12. Prokaryotic expression and characterization of the heterodimeric construction of ZnT8 and its application for autoantibodies detection in diabetes mellitus

Author

Bruno David Rovitto, Ruben F. Iacono, Silvina Noemi Valdez, Natalia Ines Faccinetti, Edgardo Poskus, Aldana Trabucchi, Luciano Lucas Guerra, Adriana Victoria Sabljic, and Silvina Sonia Bombicino

Subjects
0301 basic medicine, lcsh:QR1-502, medicine.disease_cause, Applied Microbiology and Biotechnology, Inclusion bodies, lcsh:Microbiology, law.invention, purl.org/becyt/ford/1 [https], 0302 clinical medicine, Thioredoxins, Diabetes mellitus, Autoantibody, Reticulocyte, law, AUTOANTIBODY, ZNT8, medicine.diagnostic_test, IMMUNOASSAYS, medicine.anatomical_structure, Biochemistry, Recombinant DNA, Rabbits, Thioredoxin, ZnT8, CIENCIAS NATURALES Y EXACTAS, Biotechnology, DIABETES MELLITUS, Otras Ciencias Biológicas, Recombinant Fusion Proteins, 030209 endocrinology & metabolism, Bioengineering, Enzyme-Linked Immunosorbent Assay, Zinc Transporter 8, Biology, Recombinant protein expression, Ciencias Biológicas, 03 medical and health sciences, Affinity chromatography, medicine, Escherichia coli, Animals, Humans, RECOMBINANT PROTEIN EXPRESSION, Antigens, purl.org/becyt/ford/1.6 [https], Immunoassays, Autoantibodies, Research, Fusion protein, Molecular biology, 030104 developmental biology, Diabetes Mellitus, Type 1, Immunoassay, ESCHERICHIA COLI
Abstract

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. Fil: Faccinetti, Natalia Ines. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología; Argentina Fil: Guerra, Luciano Lucas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología; Argentina Fil: Sabljic, Adriana Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología; Argentina Fil: Bombicino, Silvina Sonia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología; Argentina Fil: Rovitto, Bruno David. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología; Argentina Fil: Iacono, Ruben Francisco. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología; Argentina Fil: Poskus, Edgardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología; Argentina Fil: Trabucchi, Aldana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología; Argentina Fil: Valdez, Silvina Noemi. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología; Argentina

Published
2017

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