IDEHU   05542
INSTITUTO DE ESTUDIOS DE LA INMUNIDAD HUMORAL PROF. RICARDO A. MARGNI
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
Prokaryotic expression and immunochemical characterzation of insulinoma-associated tyrosine phosphatase 2 (IA-2) fused to thiorredoxin. Early application in the diagnostic support of autoimmune diabtes mellitus
Autor/es:
GUERRA, LUCIANO L.; FACCINETTI, NATALIA I.; TRABUCCHI, ALDANA; IACONO, RUBÉN F.; POSKUS, EDGARDO; VALDEZ, SILVINA N.
Lugar:
Mar del Plata
Reunión:
Congreso; LXII Reunión Anual Sociedad Argentina de Inmunología; 2014
Institución organizadora:
Sociedad Argentina de Inmunología
Resumen:
Prokaryotic expression and immunochemical characterzation of insulinoma-associated tyrosine phosphatase 2 (IA-2) fused to thiorredoxin. Early application in the diagnostic support of autoimmune diabtes mellitus. 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-2) was cloned into pTrxFus vector to obtain it fused to thioredoxin (TrxIA2 ic). Escherichia coli GI724 was transformed with pTrxIA-2 and cultured at 30°C. Protein expression was induced with tryptophan for 3 h; the soluble intracellular fraction was isolated and TrxIA-2 was purified by affinity chromatography. SDS-PAGE and Western Blot analysis were carried out. The ability of TrxIA-2 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-2.Quantitative competition assays with 4 IA-2A (+) patients sera were performed by adding TrxIA-2 (31 pM to 0.6 µM) to the Radioligand Binding Assay (RBA). Brigde ELISA employing TrxIA-2 and biotinylated TrxIA-2 ic, 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-2 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-2 from E. coli. We demonstrate its proper immunochemical behavior by inhibition or displacement of [35S]IA-2 binding to IA-2A and use it in early attempts for IA-2A assessment by non-radiometric immunoassays.