In the 1980s and early 1990s the principal two autoantigens recognized by ICA were identified. The first was a new isoform of glutamic acid decarboxylase (GAD65) (10
) and the second was a protein tyrosine phosphatase–like molecule (IA-2) (12
). The availability of these proteins in recombinant form allowed for the development of radioimmunoassays, which have now virtually replaced the ICA immunofluorescence test for measuring autoantibodies. A third antigen, insulin, also was identified in the 1980s (13
). This antigen is not recognized in the ICA test, which uses unfixed frozen tissue sections from which insulin and c-peptide leach out during sample preparation.
There are two isoforms of GAD, one with a molecular weight of 65,000 (GAD65) and the other with a molecular weight of 67,000 (GAD67) (14
). GAD65 is involved in the conversion of glutamic acid to γ-aminobutyric acid (GABA), a major inhibitory neurotransmitter. The two isoforms are approximately 65% identical and are expressed not only in neurons, but also in pancreatic islet cells, where GAD65 predominates. The function of GAD65 in islet cells is not known. The GAD65
gene, which is found on chromosome 10p11, encodes a protein of 585 amino acids (Table ). Autoantibodies in sera of type 1 diabetic patients are directed primarily to middle (amino acids 245–449) and C-terminal (amino acids 450–585) regions of the molecule (16
). Some GAD65 autoantibodies in diabetes sera cross-react with GAD67, the product of an unlinked but homologous gene.
Major autoantigens in type 1 diabetes
IA-2, also known as ICA512, is an unusual member of the transmembrane protein tyrosine phosphatase (PTP) family and is located on chromosome 2q35 (16
). It is atypical in that it lacks enzymatic activity because of a critical amino acid substitution at position 911 (Asp for Ala) in the catalytic domain of the molecule. The molecular weight is 106,000 and the protein is 979 amino acids in length. The protein is expressed in neuroendocrine tissues and is found in both the α and the β cells of the pancreatic islets. Immunofluorescence studies have localized IA-2 to the secretory vesicles of both endocrine and neuronal cells, but its function is not known.
A closely related protein, and another autoantigen in type 1 diabetes, is IA-2β, also known as phogrin (17
). The intracellular domain of this 111,000 molecular weight protein is 74% identical to IA-2. IA-2β, which is encoded on chromosome 7q36, carries the same amino acid substitution in its catalytic domain, and it is also expressed in neuroendocrine tissues. IA-2 and IA-2β belong to a highly conserved family of proteins with homologues in macaques, mice, rats, cows, zebra fish, Caenorhabditis elegans
, and Drosophila
). Autoantibodies to IA-2 and IA-2β are directed exclusively to their intracellular domains (16
). Most sera that recognize IA-2β also recognize IA-2, but not all sera that recognize IA-2 recognize IA-2β. For this reason IA-2 is the protein of choice for most immunoassays.
Insulin, the third autoantigen in type 1 diabetes (13
), is a short protein of 51 amino acids encoded on chromosome 11p15. As with the GAD65- and IA-2–specific antibodies (16
), autoantibodies in the sera of type 1 diabetic patients are directed primarily to conformational epitopes. In the case of insulin, these epitopes map to the B chain of human proinsulin or insulin (19
). Unlike the GAD65 and IA-2 autoantibodies, insulin autoantibodies are not useful for confirming the classification of diabetes after insulin replacement therapy has begun, because patients develop antibodies to exogenous insulin.
Extensive studies have been carried out at international workshops to standardize the assays for autoantibodies to GAD65/IA-2/insulin (20
). In the case of IA-2 and GAD65, radioligand binding assays have given precise and reproducible results. In the case of insulin, however, there has been considerable interlaboratory variation. A recently developed assay (22
) may circumvent some of the problems with the older assay and may make it possible to measure all three autoantibodies reproducibly, using small volumes of serum. Autoantibodies to other antigens also have been reported (e.g., carboxypeptidase H, ICA69, GLUT-2, SOX-13, and β cell sulfatides), but, because they are found at considerably lower frequency than autoantibodies to GAD65/IA-2/insulin, they have not been used in routine clinical studies.