Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
9267873 | Journal of Autoimmunity | 2005 | 9 Pages |
Abstract
A hybridoma secreting a human monoclonal autoantibody to the islet cell autoantigen IA-2 was prepared from peripheral lymphocytes of a patient with type 1 diabetes and Graves' disease using EBV infection followed by fusion with a mouse/human hybrid cell line. The monoclonal antibody (M13) is an IgG1/kappa and in an immunofluorescence test M13 at 1 μg/mL showed islet cell antibody reactivity equivalent to 40 JDF units. M13 IgG bound 35S-labelled IA-2 (26% at 100 μg/mL) and 125I-labelled IA-2 (34% at 100 μg/mL) in an immunoprecipitation assay and reacted well with IA-2 in western blotting analysis. Amino acids 777-808 in the PTP domain of IA-2 were found to be important for M13 binding in an analysis using modified 35S-labelled IA-2 proteins. M13 V region genes were from VH1-3, D3-22, JH4b, VKI DPK8/Vd+ and JK3 genes and showed a high replacement/silent mutation ratio for both the heavy (11.0) and the light (6.0) chain genes. Mouse monoclonal antibodies (mMAbs) reactive with at least three different epitopes within IA-2 aa 604-686 corresponding to the juxtamembrane domain were also obtained. F(abâ²)2 or Fab from the mMAbs inhibited serum IA-2 autoantibody binding to IA-2 in 20/22 diabetic sera whereas M13 F(abâ²)2 caused inhibition in only 6/22 sera. M13 is representative of some patient serum IA-2 autoantibodies and as such provides a useful tool to study autoimmune responses to IA-2.
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Authors
R. Ananieva-Jordanova, M. Evans, T. Nakamatsu, L.D.K.E. Premawardhana, J. Sanders, M. Powell, S. Chen, V. McGrath, C. Belton, C. Arnold, S. Baker, C. Betterle, R. Zanchetta, B. Rees Smith, J. Furmaniak,