Determination of glutamic acid decarboxylase antibodies (GADA) IgG subclasses - comparison of three immunoprecipitation assays (IPAs).
IgG subclasses of glutamic acid decarboxylase (GAD(65)) antibodies (GADA) may reflect the immunological state in the pancreas of GADA-positive patients with autoimmune diabetes. The use of biotin-conjugated antibodies and streptavidin Sepharose are used commonly in immunoprecipitation assays (IPA) based on (125)I- or (35)S-labelled antigens to capture IgG subclasses directed against IA-2 or GAD(65). We have compared three different immunoprecipitation assays for the determination of GADA IgG subclasses. Two of the assays were based on the biotin and streptavidin systems provided in a solid (immobilized) or liquid (mobilized) phase binding environment. The third assay was based on N-hydroxysuccinimide (immobilized) interaction with primary amines (i.e. lysine residues) on the antibody. We found the liquid phase binding assay (LPBA) to be the most stable assay, with a comparatively low coefficient of variation and background.
Department of Clinical Sciences, Lund University, Sweden. Magnus.Hillman@med.lu.se
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Hillman
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Törn
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Landin-Olsson
D000937:Antigen-Antibody Reactions; D001323:Autoantibodies / Q000097:blood*; D001710:Biotin; D003922:Diabetes Mellitus, Type 1 / Q000276:immunology*; D005968:Glutamate Decarboxylase / Q000276:immunology*; D006801:Humans; D047468:Immunoprecipitation / Q000379:methods; D007202:Indicators and Reagents; D019809:Streptavidin; D013388:Succinimides
D001323:Autoantibodies; D007202:Indicators and Reagents; D013388:Succinimides; D001710:Biotin; D019809:Streptavidin; D005968:Glutamate Decarboxylase; C001426:N-hydroxysuccinimide