Determination of glutamic acid decarboxylase antibodies (GADA) IgG subclasses - comparison of three immunoprecipitation assays (IPAs)

Clin Exp Immunol. 2007 Oct;150(1):68-74. doi: 10.1111/j.1365-2249.2007.03473.x. Epub 2007 Jul 30.

Abstract

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.

Publication types

  • Comparative Study
  • Evaluation Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antigen-Antibody Reactions
  • Autoantibodies / blood*
  • Biotin
  • Diabetes Mellitus, Type 1 / immunology*
  • Glutamate Decarboxylase / immunology*
  • Humans
  • Immunoprecipitation / methods
  • Indicators and Reagents
  • Streptavidin
  • Succinimides

Substances

  • Autoantibodies
  • Indicators and Reagents
  • Succinimides
  • Biotin
  • Streptavidin
  • Glutamate Decarboxylase
  • N-hydroxysuccinimide