Pancreatic Islet Cell Cytoplasmic Antibody in Diabetes Is Represented by Antibodies to Islet Cell Antigen 512 and Glutamic Acid Decarboxylase Mark A. Myers, Daniel U. Rabin, and Merrill J. Rowley The presence of serum islet cell cytoplasmic antibodies (ICAs) is a standard autoimmune marker for insulin- dependent diabetes mellitus (IDDM). The antigenic mol- ecule(s) responsible for ICA has not been identified, although antibodies to the 65-kDa isoform of glutamic acid decarboxylase (GAD 65 ) do contribute. We tested 129 IDDM sera for antibodies to ICA512 (anti-ICA512), anti- bodies to GAD (anti-GAD), and ICAs; we tested for inhibition of ICAs with purified recombinant ICA512 and sheep brain GAD; and we tested for immunofluorescence reactivity on COS7 cells transfected with cDNA clones encoding ICA512 and GAD 65 . The results were that anti- ICA512 antibodies contribute to ICA reactivity and that these, in combination with anti-GAD antibodies, account for most ICA reactivity in IDDM. Anti-ICA512 antibodies were present at a frequency of 51% in 61 patients with early-onset IDDM (age of onset ^20 years) of short duration (<1 month) but only in 9% of 68 patients with an onset age of >20 years and/or a disease duration of > 1 month. The frequency of anti-GAD antibodies in these sera was similar irrespective of duration or age of onset. Anti-ICA512 and anti-GAD antibodies were demonstrable by indirect immunofluorescence on transfected COS7 cells, and ICA could be inhibited using either recombi- nant ICA512 or purified brain GAD. We conclude that anti-ICA512 and anti-GAD antibodies contribute to ICA reactivity and that anti-ICA512 antibodies account for the increased frequency of ICA reactivity in early-onset IDDM of short duration. Diabetes 44:1290-1295, 1995 I nsulin-dependent diabetes mellitus (IDDM) is attribut- able to progressive autoimmune destruction of insulin- producing pancreatic islet cells reflected by autoantibodies to islet p-cell proteins in the predia- betic and diabetic stages of the disease. Antibodies to two particular autoantigenic substrates characterize the disease, the 65-kDa isozyme of glutamic acid decarboxylase (GAD 65 ) (1), and the islet cell cytoplasm (islet cell antibody [ICA]) (2), for which the identity of the responsible antigen has re- mained elusive. The presence of anti-GAD antibody and ICA From the Centre for Molecular Biology and Medicine (M.A.M., M.J.R.), Monash University, Victoria, Australia; and the Bayer Research Center (D.U.R.), West Haven, Connecticut. Address correspondence and reprint requests to Dr. Merrill J. Rowley, Centre for Molecular Biology and Medicine, Monash University, Victoria 3168, Australia. Received for publication 5 July 1995 and accepted 6 July 1995. ELISA, enzyme-linked immunosorbent assay; GAD, glutamic acid decarboxyl- ase; GST, glutathione-S-transferase; ICA, islet cell antibody; IDDM, insulin-depen- dent diabetes mellitus; JDF U, Juvenile Diabetes Foundation units; OD, optical density; PBS, phosphate-buffered saline; SM, skim milk. shows high correspondence in IDDM sera (3), and pancreatic GAD itself appears to be responsible for a proportion of ICA reactivity (4,5). On the other hand, anti-GAD antibody occurs frequently in childhood- and adult-onset types of IDDM and is persistent, whereas ICA is more associated with a child- hood onset of IDDM and is transient (6). Accordingly, there must be reactivities to one or more islet cell autoantigens other than anti-GAD that account for ICA reactivity, partic- ularly in IDDM with a young onset age. In this context, a promising new candidate is ICA512. The cDNA encoding ICA512 was isolated by antibody screening of a human islet cDNA expression library using a pool of sera from 30 patients with newly diagnosed IDDM (7). The deduced amino acid sequence of ICA512 is similar to that of protein tyrosine phosphatases, particularly the lymphocyte marker CD45. Autoantibodies to ICA512 were detected in 48% of sera from children with newly diagnosed IDDM (8). The aim of the present study was to determine the contribution made by ICA512 to the ICA reactivity of IDDM sera. Our results show that antibodies to ICA512 are most frequent in sera from juvenile patients with IDDM of recent onset and, in such patients, make a major contribution to ICA reactivity. RESEARCH DESIGN AND METHODS Sera. Sera were available from 129 patients with IDDM (mean duration of disease 2.5 years, range 0-41 years), 85 tested within 1 month of diagnosis and 44 tested more than 1 month after diagnosis. For 89 patients, the diagnosis was made before the age of 20 years (early onset), and for 40 patients, the diagnosis was made after the age of 20 years (adult onset). Control sera were from 70 healthy blood donors and 36 children with diseases other than IDDM. Details of the patients and control subjects are shown in Table 1. Collection of these sera has been described in previous publications from this laboratory (9-11). Preparation of recombinant ICA512. The intracellular portion of ICA512 (amino acids 255-548), expressed in the bacterial expression vector pGEX as a glutathione-S-transferase (GST) fusion protein, was characterized and purified as described previously (8). This GST-ICA512 fusion protein was cleaved with thrombin, and the ICA512 was sepa- rated from GST by chromatography on a glutathione-sepharose column. Detection of antibodies to ICA512 (anti-ICA512). Anti-ICA512 antibodies were measured by enzyme-linked immunosorbent assay (ELISA) using purified ICA512 as antigen. Flat-bottomed polystyrene microtiter plates (Nunc, Roskilde, Denmark) were coated overnight at 4°C with 100 \d of 1 |xg/ml ICA512 diluted in phosphate-buffered saline (PBS), pH 7.4. Plates were blocked for 2 h with 1% skim milk (SM) in PBS (pH 7.3) containing 0.05% Tween 20 (SM-PBS-Tween), then reacted with sera diluted 1:50 in SM-PBS-Tween overnight at 4°C. The plates were washed three times in SM-PBS-Tween and three times in distilled water. Bound antibodies were detected using affinity-purified alkaline phosphatase-conjugated anti-human IgG (Silenus, Hawthorn, Australia) diluted in SM-PBS without Tween 20. After 2 h at room temperature, the plates were washed as before—with SM-PBS, without Tween 20, and with distilled water—and developed using 1 mg/ml p-nitrophenyl phos- 1290 DIABETES, VOL. 44, NOVEMBER 1995 Downloaded from http://diabetesjournals.org/diabetes/article-pdf/44/11/1290/360899/44-11-1290.pdf by guest on 04 November 2022