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. 2005 Jun;140(3):564-71.
doi: 10.1111/j.1365-2249.2005.02802.x.

Epitope analysis of insulin autoantibodies using recombinant Fab

C J Padoa  1 , N J Crowther, J W Thomas, T R Hall, L M Bekris, C Torn, M Landin-Olsson, E Ortqvist, J P Palmer, A Lernmark, C S Hampe
Affiliations

Epitope analysis of insulin autoantibodies using recombinant Fab

C J Padoa et al. Clin Exp Immunol. 2005 Jun.

Abstract

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.

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Figures

Fig. 1
Fig. 1
Nucleotide sequence and deduced amino acid sequence of the light- and heavy-chain of CG7C7 monoclonal antibody (mAb). The complementarity determining regions (CDRs) according to Kabat [53] are boxed and the amino acid residue differences in comparison to the germline sequences are shown in bold type.
Fig. 2
Fig. 2
Radio-binding asay (RBA) of recombinant Fab (rFab) CG7C7 and GAD65-specific rFab N-GAD65 monoclonal antibody (mAb) to [125I]-insulin. rFab CG7C7 (black squares) and N-GAD65 mAb (white squares) at the indicated concentration were incubated with [125I]-insulin. The immunocomplexes were precipitated using Protein G Sepharose (PGS). Binding to insulin is expressed as insulin autoantibody (IAA) units.
Fig. 3
Fig. 3
Epitope analysis of CG7C7. Different concentrations of recombinant Fab (rFab) CG7C7 were used to compete insulin binding by monoclonal antibodies (mAb) mAb 1 (black triangles), mAb 125 (white diamonds) and mAbCG7C7 (black squares) at half maximal binding. Binding to insulin is expressed as a percentage, uncompeted binding is set as 100%.
Fig. 4
Fig. 4
Recombinant Fab (rFab) CG7C7 competes with the majority of sera from insulin autoantibody (IAA)-positive type 1 and type 1·5 diabetes patients. Insulin binding of serum samples of type 1 (n = 25) (a) and type 1·5 diabetes (n = 14) (b) patients in the absence (black diamonds) and presence (white diamonds) of rFab CG7C7. The data are presented on a logarithmic scale.
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