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.