1. Novel Flow Cytometric Immunoassay for Detection of Proinsulin Autoantibodies in Diabetes Mellitus Employing a Recombinant Autoantigen Expressed in E. coli
- Author
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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
0301 basic medicine ,lcsh:Immunologic diseases. Allergy ,Adult ,Male ,proinsulin ,Adolescent ,autoantibodies ,Population ,Immunology ,030209 endocrinology & metabolism ,Autoantigens ,law.invention ,Flow cytometry ,03 medical and health sciences ,Young Adult ,0302 clinical medicine ,law ,medicine ,Escherichia coli ,Immunology and Allergy ,Humans ,Multiplex ,immunoassay ,education ,Child ,Proinsulin ,Original Research ,education.field_of_study ,medicine.diagnostic_test ,Chemistry ,flow cytometry ,autoimmunity ,Autoantibody ,Infant ,Middle Aged ,Molecular biology ,Microspheres ,Recombinant Proteins ,030104 developmental biology ,Diabetes Mellitus, Type 1 ,Immunoassay ,Biotinylation ,Case-Control Studies ,Child, Preschool ,diabetes mellitus ,Recombinant DNA ,Female ,lcsh:RC581-607 ,Biomarkers - 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