Diluted patient serum (serum diluent contains sorbent to remove Rheumatoid Factor and human IgG interference) is added to wells coated with purified aCL antigen. aCL specific IgM antibody, if present, binds to the antigen. All unbound materials are washed away and the enzyme conjugate is added to bind to the antibody-antigen complex, if present. Excess enzyme conjugate is washed off and substrate is added. The plate is incubated to allow the hydrolysis of the substrate by the enzyme. The intensity of the color generated is proportional to the amount of specific IgM antibody in the sample.
Measurement of IgG, IgM and IgA cardiolipin autoantibodies (aCL) by EIA is the standard procedure for the detection of antiphospholipid antibodies (aPL) in patients with suspected antiphospholipid syndrome (APS).High aCL concentrations are associated with increased risk of venous and arterial thrombosis, recurrent pregnancy loss and thrombocytopenia. Patients with the anti-cardiolipin syndrome have one of the above clinical features and have antibodies to cardiolipin andor a positive lupus anticoagulant test. The antibodies present to cardiolipin may be of the IgG, IgA, IgM isotypes.Testing for the various antibody isotypes to cardiolipin aid in diagnosis of the anti-phospholipid syndrome in patients with SLE or lupus-like disorders. Binding of aCL to CL in patients with autoimmune diseases is dependent on the presence of the cofactor ibetai-2-glycoprotein I (ibetasub2subi-GPI) this binding is independent of ibetai-2-GPI in patients with infectious diseases (e.g., syphilis, tuberculosis). Recognition of the role of ibetai-2-GPI in the binding of aCL led to development of assay for direct measurement of ibetai-2-GPI autoantibodies using ibetai-2-GPI as antigen,allowing a clear distinction between ibetai-2-GPI autoantibodies and those that bind to CL alone.