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

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.

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-2) in Escherichia coli GI698 and GI724 strains. We have also carried out the biochemical and immunochemical characterization of TrxIA-2and design variants of non-radiometric immunoassays for the efficient detection of IA-2 autoantibodies (IA-2A).

Expression and characterization of human proinsulin fused to thioredoxin in Escherichia coli.

Native proinsulin (PI) belongs to the class of the difficult-to-express proteins in Escherichia coli. Problems mainly arise due to its high proteolytic decay and troubles to reproduce the native disulphide pattern. In the present study, human PI was produced in E.