Background: Antiphospholipid syndrome (APS) is an autoimmune disorder characterized by thrombosis and pregnancy morbidity in the presence of compatible laboratory results for antiphospholipid antibodies. Detection of anticardiolipin antibodies (aCL) by enzyme-linked immunosorbent assays (ELISA) is used to support the diagnosis of APS. aCL ELISAs suffer from poor sensitivity, specificity, and interlaboratory reproducibility. These issues lead many patients to face ambiguity in diagnosis, potentially unnecessary anticoagulation, or under-recognition of thrombotic risk. There is therefore an urgent need for improved diagnostic tools. The interlaboratory variability may stem in part from inconsistencies in the cardiolipin preparations used in the ELISA. A mixture of bovine cardiolipins is commonly used in clinical assays. Cardiolipins are a group of phospholipids consisting of a glycerol bridging 2 phosphatidyl groups, each bearing 2 fatty acyl chains. The chain length and degree of saturation of the fatty acyl chains are denoted by XX:Y, with XX representing the number of carbons in each fatty acyl chain and Y representing the number of double bonds per chain. The chemical features of cardiolipins that are required for a positive aCL ELISA result are undefined.

Aim: The aim of this study was to identify the specific chemical features of cardiolipins that are necessary for the detection of antiphospholipid antibodies in patients with APS using an immunoassay.

Methods: Bovine cardiolipin was purchased from Millipore Sigma and analyzed by gas chromatography/mass spectrometry (GC/MS). A cardiolipin library was constructed and included: 2 commercial bovine cardiolipin mixtures; 6 purified synthetic cardiolipins with defined mono-acyl chains (14:0, 16:0, 18:0, 14:1, 16:1, 18:1); 2 microbial cardiolipins, MiCL-1 from Muribaculum intestinale (18:1-16:0) and SpCL-1 from Streptococcus pyogenes (18:1-18:0), and 2 corresponding synthetic isomers with inverted chain positions (16:0-18:1 and 18:0-18:1). Patient aCL antibodies were detected by ELISA. First, 96-well plates were coated with cardiolipins. Next, plates were blocked in 10% fetal bovine serum in phosphate buffered saline. The plates were incubated with plasma from 3 APS patients with positive aCL immunoglobulin G (IgG) and 3 aCL IgG-negative controls. Horseradish peroxidase-conjugated anti-human IgG was used for detection, with 3,3′,5,5′-tetramethylbenzidine dihydrochloride substrate and 450 nm absorbance quantification.

Results: Analysis of bovine cardiolipin by GC/MS revealed that this preparation contains a variety of cardiolipin compounds, including compounds primarily with 18:2 and 18:1 fatty acyl chains but also with detectable 18:0, 16:1, 16:0, and 14:0 chains.

When tested in aCL IgG ELISA, all patient plasma samples exhibited robust IgG binding to monounsaturated cardiolipin molecular species (14:1, 16:1, 18:1 cardiolipin), whereas reactivity against cardiolipins with 4 fully saturated fatty acyl chains (14:0, 16:0, 18:0 cardiolipin) was diminished (p < 0.0001, n = 3).

All patient plasma samples exhibited strong IgG binding to the microbial cardiolipin, MiCL-1 and its chain-switched isomer. The binding was significantly higher than that to the bovine cardiolipin (p < 0.0001, n = 3). All patient plasma samples also exhibited robust binding to the other microbial cardiolipin SpCL-1, but significantly diminished binding to the chain-switched isomer (p < 0.0001, n = 3). Patient anticardiolipin IgG exhibited no significant differences in binding between the 2 commercially available bovine cardiolipin mixtures (p > 0.05).

Conclusions: Fatty acid saturation is a key determinant of cardiolipin antigenicity in APS, with no significant differences in binding due to chain length within this group of monounsaturated CLs (14:1, 16:1 and 18:1). The binding of aCL IgGs to purified microbial cardiolipins may suggest a microbial trigger that contributes to both autoantibody generation and a prothrombotic predisposition in APS. This study provides insights into the key molecular features of the phospholipid that govern detection of APS-relevant autoantibodies, offering a foundation for the development of a more specific and sensitive diagnostic assay for APS.

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2025
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