Prokaryotic expression and immunochemical characterization of insulinoma-associated tyrosine phosphatase 2 (IA-2) fused to thioredoxin. Early application in the diagnostic support of Autoimmune Diabetes Mellitus

GUERRA L; FACCINETTI N; A. TRABUCCHI; IACONO RF; POSKUS E; VALDEZ SN,

Mar del Plata

Congreso; Congreso de la Sociedad Argentina de Inmunología; 2014

Sociedad Argentina de Inmunología

Autoantibodies to IA-2 (IA-2A) are early markers of pancreatic autoimmunity, being useful in diagnostic support of Autoimmune Diabetes Mellitus. The aim of this work was to express properly folded IA-2 in order to develop non-radiometric immunoassays for IA-2A detection.The intracellular domain of IA-2 (IA-2ic) was cloned into pTrxFus vector to obtain it fused to thioredoxin (TrxIA-2ic). Escherichia coli GI724 was transformed with pTrxIA-2ic and cultured at 30°C. Protein expression was induced with tryptophan for 3 h; the soluble intracellular fraction was isolated and TrxIA-2ic was purified by affinity chromatography. SDS-PAGE and Western Blot analysis were carried out. The ability of TrxIA-2ic to compete with [35S]IA-2 was assessed qualitatively by incubating 30 IA-2A(+) patients sera with the tracer in the presence of 0.2 μM TrxIA-2ic. Quantitative competition assays with 4 IA-2A(+) patients sera were performed by adding TrxIA-2ic (31 pM to 0.6 μM) to the Radioligand Binding Assay (RBA). Brigde ELISA employing TrxIA-2ic and biotinylated TrxIA-2ic, with chemiluminescent detection, was developed using 24 IA-2A(+) sera from routine autoantibodies detection. Results were expressed as standard deviation scores (SDs).SDS-PAGE and Western blot revealed a band compatible with TrxIA-2ic expected molecular weight (~55.4 kDa) and yielded 0.72 mg of >99% pure TrxIA-2ic/L culture. All 30 IA-2A(+) sera became virtually negative under antigen excess (mean SDs changed from 17.07 to 0.34) and all dose-response curves from 4 IA-2A(+) sera showed similar protein concentration that caused 50% inhibition (53.3 to 1.08 nM). Finally, bridge ELISA showed a 79.2% sensitivity (cut off = 2 SDs) and 95% specificity.We were able to express and purify TrxIA-2ic from E. Coli. We demonstrate its proper immunochemical behaviour by inhibition or displacement of [35S]IA-2 binding to