Figure 3
Beta2-glycoprotein I (β2-GPI) dependence of immunoreactivity to cardiolipin (CL) derivatives. (a) Binding of β2-GPI to CL and its derivatives. Intriguingly, β2-GPI showed significant binding to CL, monolysocardiolipin (MCL), and dilysocardiolipin (DCL), which was comparable with that for CL at the highest concentration tested. Each data point represents the mean of triplicate determinations. (b) Box-and-whisker plot of antimonolysocardiolipin antibody (aMCL) binding in six antiphospholipid syndrome (APS) patients. Median, quartiles, range, and possibly extreme values are shown. The blocking and the washing steps were performed with foetal calf serum (FCS) (10%) in phosphate-buffered saline (PBS)-Tween-20 to provide the β2-GPI or with bovine serum albumin (BSA) (1%) or gelatine (0.5%) to avoid the presence of β2-GPI, which is commonly associated with FCS. No significant difference of aMCL reactivity was observed with different blocking solutions. (c) Thin-layer chromatography (TLC) immunostaining analysis of: lane 1: normal serum, diluted 1:100 in PBS/0.5% gelatine; lane 2: control positive aCL serum, diluted 1:100 in PBS/0.5% gelatine; lane 3: APS serum positive for both aCL and aMCL, diluted 1:100 in PBS/0.5% gelatine; lane 4: APS serum positive for both aCL and aMCL, diluted 1:1,000 in PBS/0.5% gelatine. No sera showed reactivity against DCL and hydrocardiolipin (HCL). The results in lanes 3 and 4 are representative of five different APS patients. OD, optical density; St, standard phospholipid visualisation of lipids (cardiolipin, hydrocardiolipin, monolysocardiolipin, and dilysocardiolipin) by iodide vapours.