β-2 Glycoprotein-I IgG
Anti-phospholipid antibodies (aPL) are a group of immunoglobulins associated with binding of negatively charged phospholipids including cardiolipin and phosphatidyl serine. High serum levels of anti-phospholipid antibodies are frequently found in patients with recurrent arterial and venous thromboembolism , recurrent foetal loss and thrombocytopenia, the main features of anti-phospholipid syndrome (APS). These symptoms are also often present in cases of Systemic Lupus Erythematosis (SLE), when elevated levels of aPL are detected.
It has been reported that β2-Glycoprotein I (β2-GPI), a 50 kD plasma protein, is required for binding of anti-cardiolipin and anti-phospholipid antibodies and was more recently found to be the target for a specific group of aPL. The conformational epitope for anti-β-2GPI antibodies is thought to develop when β2-GPI interacts with a lipid membrane composed of negatively charged phospholipids or, more recently reported, when it is adsorbed to the surface of a polystyrene microtitre plate.
Antibodies targeting β-2-GPI specifically have been found to be the dominant autoanitbody type in anti-phospholipid positive sera and is widely accepted as a clear diagnostic indicator of APS and of SLE associated with APS.
All 3 isotypes ( IgG, IgM and IgA) have been shown to be associated with APS. IgA anti-B2GPI in particular has been correlated significantly with venous thrombosis in these diseases. Although the levels of each isotype of B2GPI antibodies can correlate significantly with the other Isotypes, it is advised that all 3 isotypes are measured to ensure maximum sensitivity.
To increase the efficiency of clinical laboratory testing for APS, anti-cardiolipin IgM and IgG, β-2-GPI IgM and IgA ELISAs are also available from Hycor Biomedical Ltd.
The AutostatTM II 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 AutostatTM II wells are coated with purified antigen.
On adding diluted serum to the wells the antibodies present bind to the antigen. After incubating at room temperature and washing away unbound material, horseradish peroxidase conjugated anti-IgG monoclonal antibody is added, which binds to the immobilised antibodies.
Following further incubation and washing, tetra-methyl benzidine substrate (TMB) 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.