Received on September 6, 2006
Accepted on March 14, 2007

Clinical Immunology

Biosensor Analysis of 2-Glycoprotein I-Reactive Autoantibodies: Evidence for Isotype-Specific Binding and Differentiation of Pathogenic from Infection-Induced Antibodies

¹ Institute of Clinical Chemistry and Pathobiochemistry, Klinikum rechts der Isar der TU München, München, Germany
² Institute of Clinical Chemistry and Laboratory Medicine, Johannes Gutenberg-Universität Mainz, Mainz, Germany

^* To whom correspondence should be addressed. E-mail: luppa{at}klinchem.med.tum.de.

Background: For the laboratory diagnosis of the antiphospholipid syndrome (APS) we developed a biosensor with the ability to distinguish between disease-relevant anti-2-glycoprotein I (2GPI) autoantibodies (anti-2GPI) and pathogen-specific 2GPI cross-reactive antibodies that occur transiently during infections.

Methods: We used a surface plasmon resonance (SPR) biosensor device. For the detection of anti-2GPI in serum samples, affinity-purified human 2GPI was covalently attached to a functionalized n-alkanethiol self-assembling monolayer on the biosensor chip. After verifying the specificity of the biosensor system with a panel of monoclonal antibodies to 2GPI, we analyzed sera from healthy donors and patients suffering from APS, systemic lupus erythematosus (SLE), syphilis, or parvovirus B19 infections. The SPR results were compared with 2GPI-specific ELISA.

Results: Using the SPR biosensor antigen binding curves with response levels in the range of 50-500 resonance units (RU) were recorded for anti-2GPI ELISA positive APS patient sera. The amplitudes of the antiphospholipid antibody (APL) responses in the biosensor correlated with the overall IgG and IgM anti-2GPI ELISA titers with a correlation coefficient of 0.87. Moreover, we observed immunoglobulin isotype-specific association and dissociation profiles for APL binding of different APS patient sera to the biosensor-immobilized 2GPI. In contrast to APS patient samples, no significant anti-2GPI binding (response levels <35 RU) was observed in samples from healthy individuals or from patients suffering from SLE, syphilis, or parvovirus B19 infection.

Conclusions: The SPR biosensor system enables specific detection of APS-associated 2GPI-reactive APL and differentiation from 2GPI cross-reactive antibodies that occur frequently during acute infections. The established association/dissociation plot for anti-2GPI responses in APS patient sera gives additional information regarding the influence of anti-2GPI IgG and IgM isotype distribution.