The anti-GAD affinity value was expressed as the reciprocal ofKd(l/mol). == Measurement of anti-GAD65 and anti-GAD67 by immunoblotting == Recombinant GAD65 and GAD67 separated by 10% SDS Web page were transferred by electrophoresis to polyvinylidene difluoride (PVDF) membrane, blocked with 4.5% skimmed milk in Tris buffered saline pH 7.5 containing 0.1% Tween 20 (TBST) for one hour and washed three times with TBST. the affinity of binding with GAD65 and GAD67 was measured in selected keratin7 antibody sera. Sera were also tested for reactivity to GAD65 and GAD67 by immunoblotting. Of the 85 sera that contained antibodies to GAD65, 28 contained antiGAD67 measured by RIP. Inhibition with unlabeled GAD65 substantially or completely reduced antibody reactivity with both125I GAD65 and with125I GAD67. In contrast, unlabeled GAD67 reduced autoantibody reactivity with125I GAD67 but not with125I GAD65. Both populations of antibodies were of high affinity (>1010l/mol). == Conclusions == Our findings show that autoantibodies to GAD67 represent a minor population of anti-GAD65 that are reactive with a cross-reactive epitope found also on GAD67. Experimental results confirm that GAD65 is the major autoantigen in T1D, and that GAD67per sehas very low immunogenicity. We discuss our findings in light of the known similarities between the structures of the GAD Coumarin 30 isoforms, in particular the location of a minor cross-reactive epitope that could be induced by epitope spreading. == Introduction == Glutamic acid decarboxylase 65 (GAD65), a neuroendocrine enzyme, is a key autoantigen in type 1 diabetes (T1D)[1], in Latent Autoimmune Diabetes of Adults (LADA)[2]and in various neurological diseases[3],[4],[5],[6],[7]. Serum autoantibodies to GAD65 are an important marker in the early prediction and diagnosis of T1D[8],[9]. The closely related 67 kDa isoform, GAD67, is 71% identical in its amino acid sequence but is rarely an autoantigen in T1D[1],[10],[11], interacts differently with thepyridoxal-5-phosphate(PLP) co-factor, and has different kinetics for GABA synthesis in enzyme activity assays[12]. Recently, the crystal structures of human GAD65 and GAD67 were determined[13], and provided a unique insight into the structural basis for autoantigenicity of these closely related isoforms[13],[14],[15]. Analysis of the structures of the protein isoforms has allowed the identification of independent B-cell epitope clusters that locate on opposing faces of the C-terminal domains on GAD65 but not on GAD67[14]. Structural comparisons revealed two key differences between the isoforms. First, GAD65 is more flexible than GAD67, mainly at the C-terminal domains and at the catalytic loop residues. Second, there are striking differences between these isoforms in their electrostatic charge distribution[15],[16]. These structural and physicochemical differences correlate with known epitope regions in the antigenic isoform GAD65, revealing how the immunodominant epitopes on GAD65 are highly mobile and charged, relative to the corresponding regions in the non-antigenic isoform GAD67[11],[15],[16]. Although anti-GAD67 antibodies are rare, these antibodies may represent a cross-reactive population of anti-GAD65[17],[18], but this has not been formally tested. We wondered whether this cross-reactivity might reveal insights into the structural similarities between the isoforms. We therefore set out to more closely examine the reactivity of anti-GAD65 and anti-GAD67 in sera selected to contain anti-GAD65. == Methods == == Ethics statement == Human sera were originally obtained with written consent, and were derived from previous clinical and epidemiological studies on antibodies to GAD65 approved by the Monash University Human Research Ethics Committee (MUHREC). The sera had been stored without identifying information as a source of control sera to validate new anti-GAD assays, and their use for the present study was approved by MUHREC. == Sera == Eighty five stored sera that contained anti-GAD65 were selected for study. Selection was based on the availability of sufficient serum for repeat assays and the known presence of anti-GAD65 in the serum. There was a bias towards sera containing high levels of anti-GAD65, considered more likely to contain anti-GAD67, but levels of anti-GAD65 ranged from 30 to >10,000 World Health Organization (WHO) units[19],[20]. Clinical details were limited, but the patients were adults, with T1D or Latent Autoimmune Diabetes of Adults, (LADA) of Coumarin 30 varying duration. The mouse monoclonal antibody GAD6[21],[22]was used as a positive control. Ten sera known not to contain anti-GAD65 (211 WHO units) from healthy laboratory personnel were also tested, and pooled normal serum from five blood donors was included as a negative control in each assay. == Radioimmunoprecipitation assay (RIP) == Recombinant human GAD65 and GAD67 (rGAD65, rGAD67) expressed inSaccharomyces cerevisiae[23],[24]were used for all experiments. Both proteins were N-terminally truncated, and contained a hexahistidine tag at the C-terminal end to facilitate purification. However both constructs retained full enzyme activity[13], and the GAD65 construct retained all of the reactivity with diabetes sera of full length rGAD65[23],[24]. Each antigen was iodinated using chloramine T, and anti-GAD levels were measured using a standard radioimmunoprecipitation Coumarin 30 assay (RIP) previously used in antibody standardization workshops[19],[20]. In brief, 10 l of serum.
