Detection of bilayer phospholipid-binding antibodies using flow cytometry

Area of Science:

• Immunology
• Clinical Chemistry
• Biochemistry

Background:

• Antiphospholipid antibodies (APL) are typically detected using solid-phase immunoassays with cardiolipin as the capture antigen.
• Phospholipids in vivo exist as bilayers, unlike the monolayers formed in standard polystyrene assays, potentially affecting antibody detection.
• The surface density of bilayer phospholipids is significantly higher than that of monolayers.

Purpose of the Study:

• To investigate the influence of phospholipid organization on the detection of antiphospholipid antibodies (APL).
• To develop and evaluate a novel assay using bilayer phospholipids for APL detection.
• To compare the diagnostic performance of the novel assay with established ELISA methods.

Main Methods:

• Preparation of glass microspheres coated with bilayer phospholipids (cardiolipin, phosphatidylcholine, cholesterol).
• Analysis of 39 plasma samples from patients with diagnosed anticardiolipin antibodies using flow cytometry.
• Assessment of antibody binding to lipospheres, with and without apolipoprotein H, and evaluation of specificity using annexin V and liposomes.

Main Results:

• 17 out of 39 plasma samples showed positive IgG binding to the lipospheres.
• Only four additional samples tested positive upon addition of apolipoprotein H.
• Antibody binding specificity was confirmed by reversibility with annexin V and excess liposomes.

Conclusions:

• The detection of antiphospholipid antibodies using bilayer phospholipids represents an original and potentially more physiopathological approach.
• The lack of correlation between liposphere assay and ELISA results indicates that different antibody subgroups are detected by each method.
• This novel assay may improve the specificity and comprehensiveness of antiphospholipid antibody diagnostics.