Epitope analysis of insulin autoantibodies using recombinant Fab

TY - JOUR

T1 - Epitope analysis of insulin autoantibodies using recombinant Fab

AU - Padoa, C J

AU - Crowther, N J

AU - Thomas, J W

AU - Hall, T R

AU - Bekris, L M

AU - Törn, Carina

AU - Landin-Olsson, Mona

AU - Ortqvist, E

AU - Palmer, J P

AU - Lernmark, Åke

AU - Hampe, C S

PY - 2005

Y1 - 2005

N2 - 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 [I-125]-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.

AB - 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 [I-125]-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.

KW - type 1 diabetes

KW - epitopes

KW - insulin autoantibody

KW - radioligand binding

KW - assay

KW - recombinant Fab

U2 - 10.1111/j.1365-2249.2005.02802.x

DO - 10.1111/j.1365-2249.2005.02802.x

M3 - Article

VL - 140

SP - 564

EP - 571

JO - Clinical and Experimental Immunology

JF - Clinical and Experimental Immunology

SN - 0009-9104

IS - 3

ER -