Check GAD antibody positivity with the Diamyd anti-GAD RIA plate*

References1. A.Falorni, et al,Radioimmunoassays for glutamic acid decarboxylase(GAD65) and GAD65 autoantibodies using 35S or 3Hrecombinant human ligands.J.Immunol.Methods 186:89-99,19952. C.L.Vandewalle, et al,Belgian Diabetes Registry: High diagnostic sensitivityof glutamate decarboxylase autoantibodies in IDDMwith clinical onset between age 20 and 40 years.J.Clin.Endocrinol. & Metab. 80:846-851,19953. A.Falorni, et al,Diagnostic sensitivity of immunodominant epitopes ofglutamic acid decarboxylase (GAD65) autoantibodiesin childhood IDDM.Diabetologia 39:1091-98,19964. A.Falorni, et al,Autoantibody recognition of COOH-terminal epitopesof GAD65 marks the risk for insulin requirement inadult-onset diabetes mellitus. J.Clin.Endocrinol.Metab85:309-316,2000.5. L.Avesani, et al,Improved in planta expression of the human isletautoantigen glutamic acid decarboxylase (GAD65).Transgenic Research 12:203-212,20036. Marcovina et al,Evaluation of a novel radioimmunoassay using 125 I-labelled human recombinant GAD65 for the determinationof glutamic acid decarboxylase (GAD65) autoantibodiesInt J Lab Res (2000) 30: 21-26Alberto Falorni, MD, PhD is AssociateProfessor in Internal Medicine at theUniversity of Perugia, Italy. Falorni has specializedin the techniques for the study ofhumoral autoimmunity and genetics of Type1 diabetes mellitus and other endocrine autoimmunediseases. Falorni’s clinical activity isfocused on the diagnosis and treatment ofendocrine diseases. Falorni’s main scientificinterests are pathogenesis and prevention ofType 1 diabetes mellitus and of autoimmuneprimary adrenal insufficiency.LADA Diagnostics and GADTransgenic PlantsMAlberto Falorniy main research interest ispathogenesis and preventionof Type 1 diabetesmellitus and other endocrineautoimmune diseases. Ihave initially focused myattention on the role of glutamic acid decarboxylase(GAD65) as a target molecule of autoantibodies in Type1 diabetes. The development of a semi-automated procedurefor the radioimmunological determination ofGAD65 autoantibodies in human serum (1) has made itpossible to test the diagnostic sensitivity (frequency ofType 1 diabetic patients positive) and specificity (frequencyof non-diabetic subjects negative) of this immunemarker for the disease. It was shown that GAD65 autoantibodiescan be detected in over 80% of recently diagnosedType 1 diabetic patients and occur more frequentlyamong diabetic females than males. Interestingly,GAD65Ab have emerged as the immune marker athighest diagnostic sensitivity for adult-onset Type 1 diabetes(2) which has paved the way to the diagnosticuse of this marker in routine clinical practice to discriminateautoimmune from non-autoimmune cases.In the attempt of both identifying novel markersat highest diagnostic accuracy for Type 1 diabetesand elucidating the molecular mechanisms of autoantibodyformation, I have constructed chimeric molecules,generated by substitution of regions of humanGAD65 with homologous regions of GAD67 (aLeanType II diabetesLow risk forinsulin requirement-Clinical diagnosis of adult-onset diabetes mellitus28ObeseType II diabetesScreening for otherautoimmune diseasesGAD isoenzyme which is not a major diabetes-relatedautoantigen), to define the epitope regions of theautoantigen recognized by human autoantibodies (3).It was shown that human GAD65 autoantibodies areprimarily directed against epitopes located in themiddle and COOH-terminal regions of the enzymeand that levels of GAD65 antibodies specific for theCOOH-terminal end of the autoantigen discriminateType 1 diabetic children from antibody-positive childrenwho do not progress towards clinical diabetes.In addition, I have constructed a mutant form ofhuman GAD65, generated by site-directed mutagenesisof the active site of the enzyme, which has provenenzymatically inactive but immunologically indistinguishablefrom “wild-type” human GAD65.More recently, I have focused my interest on thediagnosis and clinical characteristics of the so-calledlatent autoimmune diabetes in adults (LADA). Ihave demonstrated that GAD65 antibodies can bedetected in approximately 10% of a hospital-basedpopulation of patients diagnosed with Type 2 diabeteson clinical grounds in Italy (4). AmongGAD65Ab-positive individuals, high antibody levelsand presence of antibodies directed to the COOHterminalend of the autoantigen predicted an extremelyhigh risk of progression towards insulin-dependencyand of associated organ-specific autoimmunediseases, such as thyroid autoimmune diseases orautoimmune Addison’s disease. In contrast, the presenceof low levels of GAD65 antibodies directedonly against the middle region of the enzyme discriminateda sub-population of LADA patients withclinical characteristics very similar to that of antibody-negativeType 2 patients.At present, I am testing the hypothesis that theislet autoimmune process may be modulated and theappearance of clinical signs of the disease delayed orprevented by the oral administration of recombinanthuman GAD65. To make this strategy potentiallyapplicable to clinical studies of primary prevention,we have constructed transgenic plants expressinghuman GAD65 (5), that can potentially allow us toadminister the human autoantigen without the needfor costly and time-consuming procedures of proteinpurification. We have demonstrated that, in transgenicplants, GAD65 accumulates in chloroplast tylacoidsand mitochondria and that the targeting ofhuman GAD65 to the plant cell cytosol (by substitutionof the NH 2 -terminal end of the protein with ahomologous region of GAD67) is associated with a 5-fold increase of expression levels. Ongoing studies aretesting the effect of the oral administration of transgenicplant material containing human GAD65 inanimal models of spontaneous autoimmune diabetes.page 38 dmccad june 2003