title: 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.
authors.affiliation: FIRS Laboratories, RSR Ltd, Parc Ty Glas, Llanishen, Cardiff CF14 5DU, UK.
authors.forename: N
authors.initials: N
authors.lastname: Hayakawa
authors.forename: L D K E
authors.initials: LD
authors.lastname: Premawardhana
authors.forename: M
authors.initials: M
authors.lastname: Powell
authors.forename: M
authors.initials: M
authors.lastname: Masuda
authors.forename: C
authors.initials: C
authors.lastname: Arnold
authors.forename: J
authors.initials: J
authors.lastname: Sanders
authors.forename: M
authors.initials: M
authors.lastname: Evans
authors.forename: S
authors.initials: S
authors.lastname: Chen
authors.forename: J C
authors.initials: JC
authors.lastname: Jaume
authors.forename: S
authors.initials: S
authors.lastname: Baekkeskov
authors.forename: B Rees
authors.initials: BR
authors.lastname: Smith
authors.forename: J
authors.initials: J
authors.lastname: Furmaniak
mesh_terms: D000224:Addison Disease / Q000276:immunology; D000293:Adolescent; D000328:Adult; D000368:Aged; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000097:blood / Q000235:genetics / Q000302:isolation & purification*; D000915:Antibody Affinity; D001323:Autoantibodies / Q000097:blood / Q000235:genetics / Q000302:isolation & purification*; D002648:Child; D022801:Complementarity Determining Regions / Q000235:genetics; D003922:Diabetes Mellitus, Type 1 / Q000276:immunology; D005260:Female; D005968:Glutamate Decarboxylase / Q000276:immunology*; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000097:blood / Q000235:genetics / Q000302:isolation & purification; D007074:Immunoglobulin G / Q000097:blood / Q000235:genetics / Q000302:isolation & purification; D007527:Isoenzymes / Q000276:immunology; D008297:Male; D051379:Mice; D008875:Middle Aged; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000276:immunology
chemical_list: D000911:Antibodies, Monoclonal; D001323:Autoantibodies; D022801:Complementarity Determining Regions; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D007527:Isoenzymes; D011993:Recombinant Fusion Proteins; D005968:Glutamate Decarboxylase; C402565:glutamate decarboxylase 1; C401141:glutamate decarboxylase 2