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. 2008 May 20;334(1-2):114-21.
doi: 10.1016/j.jim.2008.02.006. Epub 2008 Mar 7.

Antigen presentation of detergent-free glutamate decarboxylase (GAD65) is affected by human serum albumin as carrier protein

Jordan Steed  1 , Lisa K Gilliam, Robert A Harris, Ake Lernmark, Christiane S Hampe
Affiliations

Antigen presentation of detergent-free glutamate decarboxylase (GAD65) is affected by human serum albumin as carrier protein

Jordan Steed et al. J Immunol Methods. .

Abstract

The smaller isoform of glutamate decarboxylase (GAD65) is a major autoantigen in type 1 diabetes (TID). Its hydrophobic character requires detergent to keep the protein in solution, which complicates studies of antigen processing and presentation. In this study an attempt was made to replace detergent with human serum albumin (HSA) for in vitro antigen presentation. Different preparations of recombinant human GAD65 solubilized by HSA were incubated with Priess B cells (HLA DRB1*0401) and antigen presentation was tested with HLA DRB1*0401-restricted and epitope-specific T33.1 (GAD65 epitope 274-286) and T35 (GAD65 epitope 115-127) T-cell hybridomas. Specific epitope recognition by T33.1 (274-286) and T35 (115-127) cells varied between the different GAD65/HSA preparations, and a reverse pattern of antigen presentation was detected by the two hybridoma. The HSA-specific T-cell hybridoma 17.9 response to the different GAD65/HSA preparations followed the same pattern as that observed for the T33.1 cells. The content of immunoreactive GAD65 measured with four GAD65 antibodies indicated that the lowest GAD65 concentration resulted in the highest 274-286, but the lowest 115-127 presentation. This suggests that HSA-GAD65 interactions qualitatively affect the epitope specificity of GAD65 presentation. HSA may enhance the 274-286 epitope presentation, while suppressing the 115-127 epitope.

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Figures

Figure 1
Figure 1
T cell responses to different GAD65 preparations GAD preparations #2 (black squares), #4 (black inverted triangle), #5 (black diamond), and #3 (black triangle) were tested at the indicated concentrations with T cell hybridoma T33.1 specific to GAD65 peptide fragment 274-286 (left panel) and T cell hybridoma T35 (115-127) specific to GAD65 peptide fragment 115-127 (right panel), IL-2 responses of the T cell hybridoma is reported. IL-2 production in the presence of no antigen (black circle) is shown. The mean of four independent experiments is represented in the graph, standard error is indicated.
Figure 2
Figure 2
Different GAD65 preparations induced different T cell responses by HSA specific T cell hybridomas, indicating different HSA-concentrations. Control GAD contains only the preparation buffer without GAD65,
Figure 3
Figure 3
Different GAD preparations #4 (black diamond), #5 (black circle), #3 (black inverted triangle), and #2 (black triangle) were used in radioligand binding competition assays with monoclonal antibodies N-GAD65mAb (3a), and GAD6 (3b), human polyclonal serum 673 (3c), and human polyclonal serum 622 (3d). Competition observed in the presence of GAD buffer (black square) and BSA (white square) is shown.
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