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. 2012;7(2):e32515.
doi: 10.1371/journal.pone.0032515. Epub 2012 Feb 24.

Anti-idiotypic antibody specific to GAD65 autoantibody prevents type 1 diabetes in the NOD mouse

Xin Wang  1 , Aixia Zhang, Yu Liu, Shi Chen, Zhenqing Feng, Wenbin Shang, Marlena Maziarz, Jared Radtke, Christiane S Hampe
Affiliations

Anti-idiotypic antibody specific to GAD65 autoantibody prevents type 1 diabetes in the NOD mouse

Xin Wang et al. PLoS One. 2012.

Abstract

Overt autoantibodies to the smaller isoform of glutamate decarboxylase (GAD65Ab) are a characteristic in patients with Type 1 diabetes (T1D). Anti-idiotypic antibodies (anti-Id) directed to GAD65Ab effectively prevent the binding of GAD65 to GAD65Ab in healthy individuals. Levels of GAD65Ab-specific anti-Id are significantly lower in patients with T1D, leading to overt GAD65Ab in these patients. To determine the possible protective role of GAD65Ab-specific anti-Id in T1D pathogenesis, we developed the monoclonal anti-Id MAb 8E6G4 specifically targeting human monoclonal GAD65Ab b96.11. MAb 8E6G4 was demonstrated as a specific anti-Id directed to the antigen binding site of b96.11. MAb 8E6G4 recognized human antibodies in sera from healthy individuals, T2D patients, and T1D patients as established by ELISA. We confirmed these MAb 8E6G4-bound human antibodies to contain GAD65Ab by testing the eluted antibodies for binding to GAD65 in radioligand binding assays. These findings confirm that GAD65Ab are present in sera of individuals, who test GAD65Ab-negative in conventional detection assays. To test our hypothesis that GAD65Ab-specific anti-Id have an immune modulatory role in T1D, we injected young Non Obese Diabetic (NOD) mice with MAb 8E6G4. The animals were carefully monitored for development of T1D for 40 weeks. Infiltration of pancreatic islets by mononuclear cells (insulitis) was determined to establish the extent of an autoimmune attack on the pancreatic islets. Administration of MAb 8E6G4 significantly reduced the cumulative incidence rate of T1D and delayed the time of onset. Insulitis was significantly less severe in animals that received MAb 8E6G4 as compared to control animals. These results support our hypothesis that anti-Id specific to GAD65Ab have a protective role in T1D.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. MAb 8E6G4 binds specifically to b96.11.
A: Purified MAb 8E6G4, supernatant of hybridoma 8E6G4, or BSA were analyzed for binding by b96.11 by Dot blot. From left: supernatant of hybridoma 8E6G4, purified MAb 8E6G4, and BSA. B: Binding of MAb 8E6G4 and supernatant of hybridoma 8E6G4 to b96.11 was analyzed by immunoprecipitation followed by Western blot analysis. Binding of MAb 8E6G4 to control antibody hLF served as negative control. From left: supernatant of hybridoma 8E6G4 with b96.11, MAb 8E6G4 with b96.11, and MAb 8E6G4 with hLF. C: MAb 8E6G4 binds to b96.11 with high affinity. Dissociation constant was calculated by non-competing ELISA. Different concentrations of b96.11 were tested for binding by MAb 8E6G4. Kd of each concentration was calculated. The Kd was calculated as follows: Kd = 2(nKd′-Kd)/(n-1), where n = [b96.11]′/[b96.11]. The average Kd calculated was 480.4 pM.
Figure 2
Figure 2. MAb 8E6G4 competes with b96.11 for binding to GAD65.
A: GAD65 was translated in vitro using rabbit reticulocyte lysate. Different amounts (0–40 µl, corresponding to 0–160 ng) of recombinant GAD65 were incubated with 1.4 µg/ml MAb 8E6G4 and subsequently analyzed for binding to b96.11 by ELISA. Rabbit reticulocyte lysate alone was used as a negative control (open squares). Binding in the absence of competitor was set as 100%. B: Binding of monoclonal GAD65Ab b96.11 (black squares), b78 (open squares), and N-GAD65mAb (open circles) to radiolabeled GAD65 in the presence of the indicated concentrations of MAb 8E6G4 was determined. Binding is reported at percent binding, binding of the respective antibody to radiolabeled GAD65 in the absence of MAb 8E6G4 is set as 100%.
Figure 3
Figure 3. MAb 8E6G4 binds GAD65Ab present in human sera.
A: Human sera obtained from GAD65Ab-negative healthy individuals (circles) (n = 40), GAD65Ab-negative T2D patients (squares) (n = 22), and T1D patients (diamonds) (n = 10), were analyzed for binding to MAb 8E6G4 by ELISA. Binding of human sera to three mouse monoclonal antibodies specific to human HGF is shown as a negative control (open symbols). Median binding is indicated. B: Human sera obtained from healthy individuals (n = 40) (GAD65Ab-negative in RBA) were tested for presence of masked GAD65Ab. Sera were precipitated on immobilized MAb 8E6G4, and subsequently eluted. Elutions were tested for presence of GAD65Ab by RBA. GAD65Ab titers of sera prior to precipitation (black circles) and elutions (open circles) are presented as GAD65Ab index. Median binding is indicated.
Figure 4
Figure 4. Cumulative incidence rate of diabetes development.
NOD mice were injected with 50 and 100 µg MAb 8E6G4, presented as black circles and squares, respectively. Control animals were injected with PBS (white circles) or control mouse monoclonal antibody (white squares). Animals were weekly monitored for diabetes development. Cumulative incidence rate of diabetes development is presented as percentage.
Figure 5
Figure 5. The histopathology of pancreatic islets at onset of diabetes or at 40 weeks of age.
Representative images of pancreatic islets from animals injected with 50 µg (A) and 100 µg (B) MAb 8E6G4 IgG, or control animals (C). Pancreatic tissues were sectioned and stained with hematoxylin and eosin.
See this image and copyright information in PMC

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