Expression of recombinant glutamic acid decarboxylase in insect larvae and its application in an immunoassay for the diagnosis of autoimmune diabetes mellitus

BOMBICINO, SILVINA S.; SABLJIC, ADRIANA V.; IACONO, RUBEN F.; TRABUCCHI, ALDANA; MARFÍA, JUAN I.; GUERRA, LUCIANO L.; VALDEZ, SILVINA N.; TARGOVNIK, ALEXANDRA M.; FACCINETTI, NATALIA I.; MIRANDA, MARÍA V.

Scientific Reports

Nature

Año: 2019 vol. 9 p. 1 - 11

Autoimmune Diabetes Mellitus (DM) is a chronic disease caused by the selective destruction of insulinproducing beta cells in human pancreas. DM is characterized by the presence of autoantibodies thatbind a variety of islet-cell antigens. The 65kDa isoform of glutamate decarboxylase (GAD65) is a majorautoantigen recognized by these autoantibodies. Autoantibodies to GAD65 (GADA) are consideredpredictive markers of the disease when tested in combination with other specifc autoantibodies. Inorder to produce reliable immunochemical tests for large scale screening of autoimmune DM, largeamounts of properly folded GAD65 are needed. Herein, we report the production of human GAD65using the baculovirus expression system in two species of larvae, Rachiplusia nu and Spodopterafrugiperda. GAD65 was identifed at the expected molecular weight, properly expressed with high yieldand purity in both larvae species and presenting appropriate enzymatic activity. The immunochemicalability of recombinant GAD65 obtained from both larvae to compete with [35S]GAD65 was assessedqualitatively by incubating GADA-positive patients? sera in the presence of 1μM of the recombinantenzyme. All sera tested became virtually negative after incubation with antigen excess. Besides,radiometric quantitative competition assays with GADA-positive patients? sera were performed byadding recombinant GAD65 (0.62 nM?1.4µM). All dose response curves showed immunochemicalidentity between proteins. In addition, a bridge-ELISA for the detection of GADA was developed usingS. frugiperda-GAD65. This assay proved to have 77.3% sensitivity and 98.2% of specifcity. GAD65 couldbe expressed in insect larvae, being S. frugiperda the best choice due to its high yield and purity. Thedevelopment of a cost efective immunoassay for the detection of GADA was also aforded.