Abstract
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.
Highlights
Type 1 Diabetes Mellitus (T1DM) is a widespread disease that may lead to the development of severe clinical conditions, such as ketoacidosis, retinopathy, neuropathy, nephropathy and death due to severe metabolic imbalance
We describe the production of human GAD65 using a baculovirus expression system in two species of larvae, R. nu and S. frugiperda
Glutamic acid decarboxylase is a major autoantigen in autoimmune DM
Summary
Type 1 Diabetes Mellitus (T1DM) is a widespread disease that may lead to the development of severe clinical conditions, such as ketoacidosis, retinopathy, neuropathy, nephropathy and death due to severe metabolic imbalance. T1DM is a chronic disease caused by the selective destruction of insulin producing beta cells of the pancreas, mediated by a clinically silent autoimmune process[2,3]. A major autoantigen recognized by these autoantibodies is an islet-cell protein identified as the 65 kDa isoform of glutamic acid decarboxylase (GAD65). This enzyme catalyzes the decarboxylation of glutamic acid to γ-aminobutyric acid (GABA) and CO24–8. Native GAD65 can be produced in baculovirus-infected Sf9 cells[16,17], its expression in baculovirus-infected larvae has not been reported
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