Isolation and Characterization of Human Monoclonal Autoantibodies to Glutamic Acid Decarboxylase

Abstract

Production of human monoclonal autoantibodies to glutamic acid decarboxylase M(r) 65,000 (GAD65), characterization of their isotype, binding affinity, V region sequences and competition with autoantibodies in patients' sera is described. Lymphocytes from a patient with Addison's disease who had GAD65 autoantibodies without diabetes were immortalised and fused to a mouse/human hybridoma. In addition, mouse monoclonal antibodies to GAD65 were produced using standard techniques. F(ab')2S from our monoclonals and the GAD6 mouse monoclonal were used in competition with intact monoclonals and sera from diabetic patients for binding to 125I-labelled GAD65 (amino acids 46-586). Reactivities of the human monoclonals with GAD 65,000/67,000 M(r) chimeras were also studied. Variable region genes of human monoclonals were sequenced and analysed. The human monoclonals (n = 3) had affinity constants for GAD65 of 2.2 x 10(9), 5.8 x 10(9), 1.3 x 10(10) mol/l(-1); affinities of the mouse monoclonals (n = 5) ranged from 1.1 x 10(8) to 5.4 x 10(10) mol/l(-1). The binding of each of the human monoclonals was inhibited by GAD6 F(ab')2 and the binding of GAD6 antibody was inhibited by the human monoclonal F(ab')2S suggesting that the epitopes for these antibodies were overlapping. Studies with GAD65/GAD67 chimeras indicated that the human monoclonals reacted with C-terminal epitopes. The human monoclonals, GAD6 and 3/5 mouse monoclonals inhibited serum autoantibody binding to 125I-labelled GAD65. Overall, the human monoclonals were of high affinity, reacted with C-terminal epitopes and showed evidence of antigen driven maturation; they represented only a proportion of the repertoire of autoantibodies to GAD65 in the donor's serum and in the sera of patients with type-1 diabetes.

Recombinant Fabs of Human Monoclonal Antibodies Specific to the Middle Epitope of GAD65 Inhibit Type 1 Diabetes–Specific GAD65Abs

“…In a recent report, GAD65-specific human mAbs to the COOH-terminus of GAD65 isolated from a patient with Addison's disease partially competed GAD65 binding in 10 GAD65Ab-positive type 1 diabetic patients (31). However, it remains to be shown whether the competition is diseasespecific.…”

T lymphocyte response against pancreatic beta cell antigens in fulminant Type 1 diabetes

“…Purified recombinant human GAD65 expressed in yeast (Saccharomyces cerevisiae) was obtained from RSR (Cardiff, UK). The properties of this GAD65 preparation, including its reactivity with GAD autoantibodies (known to react with conformational epitopes on the GAD65 molecule), have previously been described in detail [13,14]. The N-terminal amino acids 2-45 were deleted from the preparation because N-terminally modified GAD65 was stable in solution.…”

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

“…Lymphocytes were isolated from the peripheral blood obtained of a 19-year-old male patient with type 1 DM and Graves' disease as described previously [12]. The patient had type 1 DM for 4 years and was taking insulin twice daily (50 Units morning and 36 Units evening of Human Mixtard 30; Novonordisk, Crawley, RH11 9RT, UK).…”

“…IgG was purified from culture supernatants by Protein A chromatography and F(ab#) 2 fragments prepared from IgG by pepsin digestion as described before [12]. IgG isotype was determined using reagents from The Binding Site (Birmingham, B29 6AT, UK) and the light chain (LC) type by western blotting using specific antibodies from Sigma (Poole, SP8 4XT, UK) [12].…”

“…IgG isotype was determined using reagents from The Binding Site (Birmingham, B29 6AT, UK) and the light chain (LC) type by western blotting using specific antibodies from Sigma (Poole, SP8 4XT, UK) [12].…”