Effect of Cardiolipin Oxidation on Solid-Phase Immunoassay for Antiphospholipid Antibodies

Michael Schlame; Ivan Haller; Lisa R. Sammaritano; Thomas J. J. Blanck

doi:10.1055/s-0037-1616751

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 2001; 86(06): 1475-1482
DOI: 10.1055/s-0037-1616751

Review Article

Schattauer GmbH

Effect of Cardiolipin Oxidation on Solid-Phase Immunoassay for Antiphospholipid Antibodies

Michael Schlame

¹Department of Anesthesiology and the Division of Rheumatic Diseases, Hospital for Special Surgery, and the Biopolymer Mass Spectrometry Core Facility, Weill Medical College of Cornell University, New York, USA

,

Ivan Haller

¹Department of Anesthesiology and the Division of Rheumatic Diseases, Hospital for Special Surgery, and the Biopolymer Mass Spectrometry Core Facility, Weill Medical College of Cornell University, New York, USA

,

Lisa R. Sammaritano

¹Department of Anesthesiology and the Division of Rheumatic Diseases, Hospital for Special Surgery, and the Biopolymer Mass Spectrometry Core Facility, Weill Medical College of Cornell University, New York, USA

,

Thomas J. J. Blanck

¹Department of Anesthesiology and the Division of Rheumatic Diseases, Hospital for Special Surgery, and the Biopolymer Mass Spectrometry Core Facility, Weill Medical College of Cornell University, New York, USA

› Author Affiliations

Abstract

Summary

Diagnostic assays for antiphospholipid antibodies are routinely performed on microtitre plates coated with cardiolipin. Here we show that contact between cardiolipin and NUNC-Immuno^® plates leads to extensive oxidation, generating a series of peroxy-cardiolipins which were identified by electrospray ionization mass spectrometry. To investigate the impact of oxidation on the antibody assay, cardiolipin was resolved into 12 molecular species, including oxidized species and non-oxidized species with different degrees of unsaturation. All 12 species reacted under anaerobic conditions with serum from patients with primary antiphospholipid syndrome. Immune reactivity was similar for tetralinoleoyl-cardiolipin, trilinoleoyl-oleoyl-cardiolipin, and peroxycardiolipins, but somewhat lower for tristearoyl-oleoyl-cardiolipin. Oxidative treatment of cardiolipin with air, cytochrome c, or Cu²⁺/tert-butylhydroperoxide, either before or during the assay, did not enhance immune reactivity. Similar results were obtained with a monoclonal IgM from lupus-prone mice, that binds cardiolipin in the absence of protein cofactors. We conclude that the solid-phase assay for antiphospholipid antibodies can be supported by various oxidized and nonoxididized molecular species of cardiolipin.

Keywords

Anticardiolipin antibodies - antigen specificity - antiphospholipid syndrome - enzyme-linked immunoassay - lipid oxidation

Full Text

References

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