Epitope analysis of insulin autoantibodies using recombinant Fab.
Autoantibodies to insulin are often the first autoantibodies detected in young children with type 1 diabetes and can be present before the onset of clinical diabetes. These autoantibodies and their epitopes are, however, not well characterized. We explored the use of monoclonal antibodies and their recombinant Fab as reagents for epitope analysis. In this study we cloned and characterized the recombinant Fab of the insulin-specific monoclonal antibody CG7C7. We found the epitope of this antibody to be located predominantly at the A-chain loop of the insulin molecule. The recombinant Fab was then used to compete for insulin binding against insulin autoantibodies present in sera from patients with type 1 or type 1.5 diabetes. In competition experiments with sera positive for autoantibodies to insulin the recombinant Fab significantly reduced the binding to [125I]-insulin by sera of type 1 (n = 35) and type 1.5 diabetes [latent autoimmune diabetes in adults (LADA)] (n = 14) patients (P < 0.0001). We conclude that competition between insulin-specific monoclonal antibodies or their recombinant Fab and insulin autoantibodies should prove useful in the epitope analysis of autoantibodies to insulin.
Padoa
C J
CJ
Department of Chemical Pathology, University of the Witwatersrand and National Health Laboratory Service, Johannesburg, South Africa.
Crowther
N J
NJ
Thomas
J W
JW
Hall
T R
TR
Bekris
L M
LM
Torn
C
C
Landin-Olsson
M
M
Ortqvist
E
E
Palmer
J P
JP
Lernmark
A
A
Hampe
C S
CS
D000293:Adolescent; D000328:Adult; D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology*; D001323:Autoantibodies / Q000276:immunology*; D001483:Base Sequence; D002648:Child; D002675:Child, Preschool; D003922:Diabetes Mellitus, Type 1 / Q000276:immunology*; D018604:Epitope Mapping / Q000379:methods; D000939:Epitopes / Q000276:immunology*; D005260:Female; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000276:immunology*; D007223:Infant; D007328:Insulin / Q000276:immunology; D007330:Insulin Antibodies / Q000276:immunology*; D008297:Male; D011236:Prediabetic State / Q000276:immunology*; D011994:Recombinant Proteins / Q000276:immunology
D000911:Antibodies, Monoclonal; D001323:Autoantibodies; D000939:Epitopes; D007140:Immunoglobulin Fab Fragments; D007328:Insulin; D007330:Insulin Antibodies; D011994:Recombinant Proteins