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

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.

Novel bridge multi-species ELISA for detection of SARS-CoV-2 antibodies.

Considering the course of the current SARS-CoV-2 pandemic, it is important to have serological tests for monitoring humoral immune response against SARS-CoV-2 infection and vaccination. Herein we describe a novel bridge enzyme-linked immunosorbent assay (b-ELISA) for SARS-CoV-2 antibodies detection in human and other species, employing recombinant Spike protein as a unique antigen, which is produced at high scale in insect larvae. METHODS: Eighty two human control sera/plasmas and 169 COVID-19 patients' sera/plasmas, confirmed by rRT-PCR, were analyzed by the b-ELISA assay. In addition, a total of 27 animal sera (5 horses, 13 rats, 2 cats and 7 dogs) were employed in order to evaluate the b-ELISA in other animal species. RESULTS: Out of the 169 patient samples, 129 were positive for IgG anti-SARS-CoV-2 and 40 were negative when they were tested by ELISA COVIDAR® IgG. When a cut-off value of 5.0 SDs was established, 124 out of the 129 COVID-19 positive samples were also positive by our developed b-ELISA (sensitivity: 96.12%). Moreover, the test was able to evaluate the humoral immune response in animal models and also detected as positive a naturally infected cat and two dogs with symptoms, whose owners had suffered the COVID-19 disease. CONCLUSION: The obtained results demonstrate that the method developed herein is versatile, as it is able to detect antibodies against SARS-CoV-2 in different animal species without the need to perform and optimize a new assay for each species.

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

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.

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

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.

Novel prokaryotic expression of thioredoxin-fused insulinoma associated protein tyrosine phosphatase 2 (IA-2), its characterization and immunodiagnostic application.

BACKGROUND: The insulinoma associated protein tyrosine phosphatase 2 (IA-2) is one of the immunodominant autoantigens involved in the autoimmune attack to the beta-cell in Type 1 Diabetes Mellitus. In this work we have developed a complete and original process for the production and recovery of the properly folded intracellular domain of IA-2 fused to thioredoxin (TrxIA-2ic) in Escherichia coli GI698 and GI724 strains. We have also carried out the biochemical and immunochemical characterization of TrxIA-2icand design variants of non-radiometric immunoassays for the efficient detection of IA-2 autoantibodies (IA-2A). RESULTS: The main findings can be summarized in the following statements: i) TrxIA-2ic expression after 3 h of induction on GI724 strain yielded ≈ 10 mg of highly pure TrxIA-2ic/L of culture medium by a single step purification by affinity chromatography, ii) the molecular weight of TrxIA-2ic (55,358 Da) could be estimated by SDS-PAGE, size exclusion chromatography and mass spectrometry, iii) TrxIA-2ic was properly identified by western blot and mass spectrometric analysis of proteolytic digestions (63.25 % total coverage), iv) excellent immunochemical behavior of properly folded full TrxIA-2ic was legitimized by inhibition or displacement of [35S]IA-2 binding from IA-2A present in Argentinian Type 1 Diabetic patients, v) great stability over time was found under proper storage conditions and vi) low cost and environmentally harmless ELISA methods for IA-2A assessment were developed, with colorimetric or chemiluminescent detection. CONCLUSIONS: E. coli GI724 strain emerged as a handy source of recombinant IA-2ic, achieving high levels of expression as a thioredoxin fusion protein, adequately validated and applicable to the development of innovative and cost-effective immunoassays for IA-2A detection in most laboratories.

Detección y caracterización de autoanticuerpos anti-ZnT8 en pacientes argentinos con diabetes mellitus tipo 1 / Detection and characterisation of zinc transporter 8 autoantibody in Argentinian patients with type 1 diabetes mellitus

Introducción y objetivos: El transportador de Zn8 (ZnT8), identificado como el cuarto autoantígeno inmunodominante en la diabetes mellitus (DM) tipo 1, presenta un polimorfismo en el residuo 325 (Arg/Trp). Sin embargo, aún no se encuentra suficientemente definido el papel de este residuo en la inmunorreactividad de los autoanticuerpos anti-ZnT8 (ZnT8A). En este contexto, el objetivo del presente trabajo involucró la detección y caracterización inmuno-química de los ZnT8A en DM tipo 1 empleando diferentes variantes antigénicas de ZnT8 con Arg o Trp en la posición 325, o bien construcciones quiméricas con ambos aminoácidos en dicha posición. Materiales y métodos: Se evaluaron 100 sueros de pacientes argentinos con reciente diagnóstico de DM tipo 1. La determinación de ZnT8A se realizó mediante el ensayo de unión de radioligando, utilizando distintas variantes antigénicas: ZnT8-Arg325, ZnT8-Trp325, ZnT8- Arg-Trp325, ZnT8-Arg-Arg325 y ZnT8-Trp-Trp325. Paralelamente se determinó la presencia de los otros marcadores de autoinmunidad para DM por ensayo de unión de radioligando. Resultados: De los 100 pacientes estudiados, 65 fueron ZnT8A+ para al menos alguna de las variantes antigénicas empleadas. Ocho reconocieron todas las formas recombinantes de ZnT8. La mayoría (56) resultaron positivos para el heterodímero (ZnT8-Arg-Trp325), 25 de los cuales reconocieron además el homodímero ZnT8-Arg-Arg325 y el monómero ZnT8-Arg325. Nueve pacientes presentaron únicamente ZnT8A como marcador de autoinmunidad. Por otro lado, los niveles de señales obtenidos con el heterodímero fueron significativamente mayores a los niveles alcanzados empleando cualquiera de las otras variantes antigénicas (mediana 17,99 vs. 10,71; 6,32; 5,66 y 4,44 SD scores para ZnT8-Arg325, ZnT8-Trp325, ZnT8-Arg-Arg325 y ZnT8-Trp-Trp325, respectivamente; p < 0,05, test t de Mann-Whitney). Conclusiones: Se logró caracterizar inmunoquímicamente los ZnT8A presentes en pacientes argentinos con DM tipo 1 empleando diferentes variantes antigénicas de ZnT8. Se observó que la incorporación de ZnT8A, en combinación con los marcadores clásicos, incrementa la sensibilidad diagnóstica de autoinmunidad. La reactividad exclusiva de ZnT8A por las variantes antigénicas que contienen arginina en el residuo 325 evidencia la existencia de epítopes dependientes del aminoácido presente en dicho residuo. Además, la aplicación de variantes diméricas reveló la existencia de epítopes definidos por la estructura cuaternaria de ZnT8. La construcción heterodimérica fue la que mostró la mejor combinación de sensibilidad y especificidad a los fines del screening rutinario de ZnT8A.

Introduction and objectives: The aim of this study involved the detection and immunochemical characterisation of ZnT8 autoantibodies (ZnT8A) in new-onset type 1 Argentinian diabetic patients. Material and methods: One hundred sera from type 1 diabetic patients were tested for ZnT8A. The antigens employed were obtained using cDNA plasmids encoding the C-terminal domains of ZnT8 carrying 325Arg, 325Trp and 3 dimeric constructs (ZnT8-Arg-Trp325, ZnT8- Arg-Arg325 and ZnT8-Trp-Trp325). ZnT8A were assessed by radioligand binding assay (RBA). Other islet-autoantibodies were also tested by RBA. Results: Among the 100 type 1 diabetic patients, the prevalence of ZnT8A was 65.0%, 8 recognized all recombinant forms of ZnT8. Most patients (56) were positive for the heterodimer (ZnT8-Arg-Trp325), being 25 of them also positive for the homodimer ZnT8-Arg-Arg325 and monomer ZnT8-Arg325. Single reactivity against ZnT8A was found in 9.0% of the group. Besides, the highest signal values were obtained with the heterodimeric variant (median 17.99 vs. 10.71, 6.32, 5.66 and 4.44 SD scores for ZnT8-Arg325, ZnT8-Trp325, ZnT8-Arg-Arg325 y ZnT8-Trp-Trp325, respectively; p < 0.05, Mann-Whitney t test). Conclusion: Immunochemical characterisation of ZnT8A present in type 1 Argentinian diabetic patients was accomplished, employing different variants of ZnT8. An increased detection of autoimmunity was found when ZnT8A was employed in combination with the other islet-autoantibodies. The presence of autoantibodies that recognized only constructions containing Arg reveals the existence of epitopes dependent of the amino acid present at residue 325. Furthermore, application of dimeric constructions revealed the existence of quaternary structure-defined epitopes which were recognized by type 1 diabetic patients. Finally, the highest combined sensitivity and specificity for routine screening of ZnT8A was accomplished with the heterodimeric construction.