info:eu-repo/semantics/article
Expression of recombinant glutamic acid decarboxylase in insect larvae and its application in an immunoassay for the diagnosis of autoimmune diabetes mellitus
Fecha
2019-01Registro en:
Trabucchi, Aldana; Bombicino, Silvina Sonia; Targovnik, Alexandra Marisa; Marfía, Juan Ignacio; Sabljic, Adriana Victoria; et al.; Expression of recombinant glutamic acid decarboxylase in insect larvae and its application in an immunoassay for the diagnosis of autoimmune diabetes mellitus; Nature Publishing Group; Scientific Reports; 9; 1; 1-2019; 1-11
2045-2322
CONICET Digital
CONICET
Autor
Trabucchi, Aldana
Bombicino, Silvina Sonia
Targovnik, Alexandra Marisa
Marfía, Juan Ignacio
Sabljic, Adriana Victoria
Faccinetti, Natalia Ines
Guerra, Luciano Lucas
Iacono, Ruben Francisco
Miranda, Maria Victoria
Valdez, Silvina Noemi
Resumen
Autoimmune Diabetes Mellitus (DM) is a chronic disease caused by the selective destruction of insulin producing beta cells in human pancreas. DM is characterized by the presence of autoantibodies that bind a variety of islet-cell antigens. The 65 kDa isoform of glutamate decarboxylase (GAD65) is a major autoantigen recognized by these autoantibodies. Autoantibodies to GAD65 (GADA) are considered predictive markers of the disease when tested in combination with other specific autoantibodies. In order to produce reliable immunochemical tests for large scale screening of autoimmune DM, large amounts of properly folded GAD65 are needed. Herein, we report the production of human GAD65 using the baculovirus expression system in two species of larvae, Rachiplusia nu and Spodoptera frugiperda. GAD65 was identified at the expected molecular weight, properly expressed with high yield and purity in both larvae species and presenting appropriate enzymatic activity. The immunochemical ability of recombinant GAD65 obtained from both larvae to compete with [35S]GAD65 was assessed qualitatively by incubating GADA-positive patients’ sera in the presence of 1 μM of the recombinant enzyme. All sera tested became virtually negative after incubation with antigen excess. Besides, radiometric quantitative competition assays with GADA-positive patients’ sera were performed by adding recombinant GAD65 (0.62 nM–1.4 µM). All dose response curves showed immunochemical identity between proteins. In addition, a bridge-ELISA for the detection of GADA was developed using S. frugiperda-GAD65. This assay proved to have 77.3% sensitivity and 98.2% of specificity. GAD65 could be expressed in insect larvae, being S. frugiperda the best choice due to its high yield and purity. The development of a cost effective immunoassay for the detection of GADA was also afforded.