The lack of anti-idiotypic antibodies, not the presence of the corresponding autoantibodies to glutamate decarboxylase, defines type 1 diabetes

Abstract

Autoantibodies to glutamate decarboxylase 65 (GAD65Ab) are commonly believed to be a major characteristic for type 1 diabetes (T1D). We investigated the presence of GAD65Ab in healthy individuals (n = 238) and first-degree relatives (FDRs) of T1D patients (n = 27) who tested negative for GAD65Ab in conventional RIAs. Sera were applied to affinity columns coated with GAD65-specific mAbs to absorb anti-idiotypic antibodies (anti-Ids). The absorbed sera were analyzed for binding to GAD65 by RIAs. Both healthy individuals and FDRs present GAD65Ab that are inhibited by anti-Id, masking them in conventional detection methods. The presence of GAD65Ab-specific anti-Ids was confirmed by competitive ELISA. Remarkably, T1D patients (n = 54) and Stiff Person Syndrome patients (n = 8) show a specific lack of anti-Ids to disease-associated GAD65Ab epitopes. Purified anti-Ids from healthy individuals and FDRs inhibited the binding of GAD65Ab from T1D patients to GAD65. We conclude that masked GAD65Ab are present in the healthy population and that a lack of particular anti-Ids, rather than GAD65Ab per se, is a characteristic of T1D. The lack of these inhibitory antibodies may contribute to T cell activation by GAD65Ab.

Fig. 1.

GAD65Ab in healthy individuals and FDRs are revealed upon removal of inhibitor. (a) Sera of healthy individuals and FDRs contain GAD65Ab/inhibitor complexes, whereas T1D patients and SPS patients lack inhibitors to disease-specific GAD65Ab. Sera of healthy individuals (A) and FDRs (B) that tested negative for GAD65Ab in conventional RIA and sera from GAD65Ab-positive T1D patients (C) and SPS patients (D) were absorbed on immobilized b96.11-PAS and b78-PAS. The serum samples and the flow-through of beads were tested for the presence of GAD65Ab in an RIA. Binding to GAD65 before (circles) and after absorption to b96.11 (triangles) or b78 (squares) is shown in cpm. SPS patients that are also diagnosed with T1D are represented by the filled symbols. Median binding is indicated. (b) ELISA of isolated inhibitor from a healthy individual. Binding of inhibitor isolated from b96.11-PAS or b78-PAS after absorption with serum from a healthy individual to b96.11-HRP (triangles) and b78-HRP (squares) was analyzed in the presence of the indicated concentrations of human recombinant GAD65. Binding is shown as percent binding with binding in the absence of GAD65 set as 100%

Fig. 2.

The inhibitor is specific to GAD65Ab. (a) GAD65 binding by unmasked sera is competed by recombinant human GAD65 and not by BSA. Sera of FDR absorbed by b96.11-PAS were tested for binding to ^[35]S-GAD65 in the presence of recombinant human GAD65 (150 mM) (○), or BSA (150 nM) (◇). Median binding is indicated. (b) Absorption of inhibitors that reduce GAD65Ab binding is specific to GAD65Ab and cannot be achieved by an irrelevant human mAb. Sera of healthy individuals (n = 15) and FDR (n = 15) were absorbed to immobilized HAA1. The flow-through was tested for binding to GAD65. GAD65Ab titer before (○) and after (●) absorption is shown in cpm. Median binding is indicated.

Fig. 3.

GAD65Ab and inhibitors are present only in the sera's Ig fraction. (a) Purified Ig and depleted serum were prepared from four FDRs (A–D). Absorbed sera (empty bars), purified Ig before (gray bars) and after absorption (black bars), Ig-depleted sera before (bars with horizontal lines) and after absorption (bars with vertical lines) were tested for binding to GAD65. The data show that affinity purification of GAD65Ab-negative sera on b96.11-PAS leads to the dissociation of the immune complexes to reveal the presence of hitherto undetectable GAD65Ab. (b) The above-described Ig-depleted serum fractions (Left) and purified Ig fractions (Right) were tested with goat-anti human κ chain. (Center) Molecular weight markers are shown.

Fig. 4.

Anti-Ids are cross-inhibitory between healthy individuals and FDRs and inhibit GAD65 binding by T1D patients' sera. (a) Anti-Ids were isolated from a FDR as described and added to the unmasked serum at the indicated concentrations. GAD65 binding is shown as percent binding (binding of absorbed, uninhibited serum is set to 100%). A dose-dependent inhibition of GAD65 binding was observed. (b) Anti-Ids were isolated from a healthy individual as described. Anti-Ids (20 μl) from the healthy individual (filled bars) and the FDR (bars with horizontal lines) were added to unmasked sera of a healthy individual (A), FDR (B), and unabsorbed sera of four T1D patients (C_1–4). GAD65 binding is reported in cpm.