Tissue Transglutaminase IgA
Enzyme-linked immunosorbent assay method for the semi-quantitative determination of specific IgA autoantibodies to tTg in human serum.
Coeliac Disease is a form of gluten sensitive enteropathy (GSE), characterised by chronic inflammation of the intestinal mucosa and villous atrophy. It is a relatively common disease which can affect between one in two thousand to one in three hundred of the population depending on the country. Patients with celiac disease may suffer from diarrhoea, other gastrointestinal problems, anaemia and fatigue.
The European Society of Paediatric Gastroenterology and Nutrition (ESPGHAN) have defined the following criteria for rapid diagnosis of the disease: a) a single positive gut biopsy, b) at least two of the following positive sera tests; IgG/IgA anti-gliadin antibodies, IgA anti-endomysial antibodies (EMA) and R1 anti-reticulin antibodies.
Recent reports indicate that the protein cross-linking enzyme, tissue transglutaminase (tTg), is the antigenic target for anti-EMA and the predominant autoantigen in coeliac disease. Detection of anti-tTg antibodies is therefore regarded as an important, reliable, non-invasive tool in the diagnosis of celiac disease in both children and adults.
The AutostatII assay for detection of autoantibodies is a solid phase immunosorbent assay (ELISA) in which the analyte is indicated by a colour reaction of an enzyme and substrate. The AutostatII wells are coated with purified antigen (1).
On adding diluted serum to the wells the antibodies (2) present bind to the antigen. After incubating at room temperature and washing away unbound material, horseradish peroxidase conjugated anti-IgA antibody (3) is added, which binds to the immobilised antibodies.
Following further incubation and washing, tetra-methyl benzidine substrate (TMB) (4) is added to each well. The presence of the antigen-antibody-conjugate complex turns the substrate to a dark blue colour. Addition of the stop solution turns the colour to yellow.
The colour intensity is proportional to the amount of autoantibodies present in the original serum sample.