Recombinant Fabs of human monoclonal antibodies specific to the middle epitope of GAD65 inhibit type 1 diabetes-specific GAD65Abs.
Autoantibodies to the 65-kDa isoform of GAD (GAD65Abs) are associated with type 1 diabetes development, but the conformational nature of the GAD65Ab epitopes complicates the evaluation of disease risk. Six GAD65-specific recombinant Fabs (rFabs) were cloned from monoclonal antibodies b96.11, DP-C, DP-A, DPD, 144, and 221-442. The binding of GAD65Abs in 61 type 1 diabetic patients to GAD65 was analyzed by competitive radioimmunoassays with the six rFabs to ascertain disease-specific GAD65Ab binding specificities. The median binding was reduced significantly by rFab b96.11 (72%) (P < 0.0001), DP-A (84%) (P < 0.0001), DP-C (84%) (P < 0.0001), 221-442 (79%) (P < 0.0001), and DP-D (80%) (P < 0.0001). The competition pattern in type 1 diabetic patients differed from that in GAD65Ab-positive late autoimmune diabetes in adults (LADA) patients (n = 44), first-degree relatives (n = 38), and healthy individuals (n = 14). Whereas 87 and 72% of the type 1 diabetic sera were competed by rFab b96.11 and DP-C, respectively, only 34 and 26% of LADA patients, 18 and 25% of first-degree relatives, and 7 and 28% of healthy individuals showed competition (P < 0.0001). These findings support the view that type 1 diabetes is associated with disease- and epitope-specific GAD65Abs and supports the notion that the middle epitope is disease associated. These GAD65-specific rFabs should prove useful in predicting type 1 diabetes and in the study of conformational GAD65Ab epitopes.
Padoa
Carolyn J
CJ
Department of Medicine, University of Washington, Seattle 98195, USA.
Banga
J Paul
JP
Madec
Anne-Marie
AM
Ziegler
Manfred
M
Schlosser
Michael
M
Ortqvist
Eva
E
Kockum
Ingrid
I
Palmer
Jerry
J
Rolandsson
Olov
O
Binder
Katherine A
KA
Foote
Jefferson
J
Luo
Dong
D
Hampe
Christiane S
CS
D000293:Adolescent; D000328:Adult; D000368:Aged; D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity; D001323:Autoantibodies / Q000276:immunology*; D002648:Child; D003922:Diabetes Mellitus, Type 1 / Q000276:immunology*; D005968:Glutamate Decarboxylase / Q000037:antagonists & inhibitors / Q000276:immunology*; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry; D007147:Immunoglobulin Light Chains / Q000737:chemistry; D007135:Immunoglobulin Variable Region / Q000737:chemistry; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000276:immunology*; D008875:Middle Aged; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000276:immunology; D012016:Reference Values; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D000911:Antibodies, Monoclonal; D001323:Autoantibodies; D007140:Immunoglobulin Fab Fragments; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D007527:Isoenzymes; D011994:Recombinant Proteins; D005968:Glutamate Decarboxylase; C401141:glutamate decarboxylase 2