Rheumatoid Factor IgG
Enzyme-linked immunosorbent assay method for the quantitative determination of specific IgG Rheumatoid Factor in human serum.
Immunoglobulins which bind to the Fc region of IgG (rheumatoid factors) are characteristically present in the serum of rheumatoid arthritis patients, although they may also be found at lower frequency in certain other diseases. Initial tests for rheumatoid factor (RF) were based on agglutination of IgG-coated red blood cells or latex particles, but, because these methods detected predominantly IgM RF, the presence of rheumatoid factors of other antibody classes (which are much poorer agglutinins) was not recognised for some time. IgG, IgA, IgD and IgE rheumatoid factors have now been identified.
Evidence for the existence of IgG RF first came from ultracentrifugation studies, and since then a number of methods for its detection have been developed. These include radioimmunoassay, immunofluorescence assay and, more recently, ELISA. Animal IgG or the isolated Fc region of human IgG are generally used as antigen.
Estimates of the frequency of IgG RF in RA patients have varied from below 50% to over 90%, and although it is usually found in association with IgM RF, it can occasionally occur alone, that is, in so-called seronegative patients. IgG RF is rare in juvenile RA but can be detected in some patients with other connective tissue diseases and bacterial endocarditis. It also occurs at low frequency and at relatively low level in normal healthy individuals.
High levels of IgM RF are associated with a poor prognosis but changes in the level of this class do not correspond to disease activity. However, IgG RF in serum (and in synovial fluid) has been shown to correlate with the severity of disease. Very high levels of IgG RF in RA patients have also been found to have a strong association with vasculitis and a poor prognosis. Thus a test for this class of RF may be of greater use in patient management than simple agglutination assays which are sensitive only to IgM class antibodies.
The AutostatII IgG Rheumatoid Factor ELISA uses rabbit IgG as antigen. The monoclonal antibody-enzyme conjugate employed as a second antibody recognises all the IgG subclasses, making this quantitative test highly specific and sensitive.
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-IgG monoclonal 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.