Isolation and characterisation of a human monoclonal autoantibody to the islet cell autoantigen IA-2

A hybridoma secreting a human monoclonal autoantibody to the islet cell autoantigen IA-2 was prepared from peripheral lymphocytes of a patient with type I diabetes and Graves' disease using EBV infection followed by fusion with a mouse/human hybrid cell line. The monoclonal antibody (M 13) is an IgG1/kappa and in an immunofluorescence test M 13 at I mu g/mL showed islet cell antibody reactivity equivalent to 40 JDF units. M13 IgG bound 35 S-labelled IA-2 (26% at 100 mu g/mL) and 121 I-labelled IA-2 (34% at 100 mu g/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 M 13 binding in an analysis using modified 35 S-labelled IA-2 proteins. M13 V region genes were from VHI-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(ab')2 or Fab from the mMAbs inhibited serum IA-2 autoantibody binding to IA-2 in 20/22 diabetic sera whereas M13 F(ab')2 caused inhibition in only 6/22 sera. M 13 is representative of some patient serum TA-2 autoantibodies and as such provides a useful tool to study autoimmune responses to IA-2. (c) 2005 Elsevier Ltd. All rights reserved.