Abstract
The autoimmune disease antiphospholipid syndrome (APS) is characterized by the presence of anticardiolipin antibodies (aCL), along with anti-β2-glycoprotein I (β2GPI) antibodies and lupus anticoagulant (LA). In this study, we developed a time-resolved fluoroimmunoassay (TRFIA) system for simultaneous quantification of aCL IgG and IgM. A 96-well microtiter plate precoated with the complex of cardiolipin from bovine heart and bovine β2GPI was incubated with the anticardiolipin IgG and IgM standard substance or serum, and the conjugate of Eu3+-labeled anti-human IgG and Sm3+-labeled anti-human IgM was pipetted to the wells to form a tipical double-antibody-sandwich immunoreactions; finally the fluorescent intensity of Eu3+ and Sm3+ was detected to reflect the quantity of anticardiolipin IgG and IgM. This assay showed a good relationship between fluorescence intensities and the concentration of anticardiolipin antibody(aCL) IgG and IgM, with a low-end sensitivity of 0.1 U/ml for IgG and 0.1 U/ml for IgM, respectively. The intra- and inter-assay coefficients of variation (CV) of the calibrators was 3.0% and 4.51% for IgG, and 2.76% and 4.45% for IgM. The average recovery was 100.38% for aCL IgG and 100.45% for aCL IgM. For serum samples, the results of our method showed a good correlation with those obtained with ELISA kit. Simultaneous detection of aCL-IgG and aCL-IgM in the same reaction well can optimize assay performance by avoiding potential influence of different reaction conditions-timing, and well-to-well difference in concentration and characteristics of cardiolipin antigen. The results of a combo aCL-IgG and aCL-IgM assay for the same sample are more consistent and more reliable. This dual-label time-resolved fluoroimmunoassay is sensitive for detecting aCL IgG and IgM across a wide concentration range with stable reagents and may assist in the clinical diagnosis of antiphospholipid syndrome.
Highlights
Anticardiolipin antibodies are autoimmune antibodies that target the negatively charged cardiolipin on the platelet and the cytomembrane of the endotheliocyte
Graham Hughes and his team published their first report that showed an association of aCL with venous thrombosis, recurrent pregnancy loss, thrombocytopenia, and pulmonary hypertension, which are associated with antiphospholipid syndrome (APS) [2,3]
APS still remains a diagnostic challenge for clinicians, largely due to issues related to laboratory testing as well as the expanding range of reported clinical manifestations of APS
Summary
Anticardiolipin antibodies (aCL) are autoimmune antibodies that target the negatively charged cardiolipin on the platelet and the cytomembrane of the endotheliocyte. They are the main antiphospholipid antibodies (aPL), along with anti-β2-glycoprotein I (β2GPI) antibodies and lupus anticoagulant (LA), that characterize the autoimmune disease antiphospholipid syndrome (APS). Employing active macromolecular enzyme as maker, the aCL ELISA itself has methodological and diagnostic limitations, its limited sensitivity, restricted linear range and poor measurement accuracy, which may make it fail to pick up the underlying patients and contribute to the explanation of ‘‘seronegative APS”(a case that patients do have clinical signs suggestive of APS but persistently test negative for antiphospholipid antibodies) [9]
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