Anticardiolipin (aCL) and anti-beta 2-glycoprotein I(anti beta 2GPI) antibodies have been shown in animal models as not cross-reacting antibody populations. This observation prompted us to prove if anti-beta 2GPI exist in human sera by using a reliable method and then to investigate if these are independent from aCl antibodies. We have developed a new ELISA for the detection of anti-beta 2GPI antibodies employing the coating of the protein in carbonate buffer to irradiated microtitre plates and the filtration of serum samples, that makes irrelevant the binding to the uncoated wells. IgG F(ab)2 fragments from IgG positive sera were shown bind beta 2GPI, providing that the binding was a specific antibody binding, mediated by the antigen binding site of the antibody molecule: moreover the antibodies were not able to differentiate native and delipidated beta 2GPI coated plates, making a possible role of a phospholipid contaminant unlikely. On the other hand, the phosphorus content of native as well as delipitated beta 2GPI was undetectable. IgG, but not IgM, anti-beta 2GPI antibodies were classically inhibited by the addition of soluble beta 2GPI, while cardiolipin liposomes appear to modify the reaction in a completely different way, possibly by the described interaction between cardiolipin and beta 2GPI.(ABSTRACT TRUNCATED AT 250 WORDS)
SummaryLupus anticoagulant (LA) is a general term to define immunoglobulins interfering with phospholipid-dependent coagulation tests. It is now clear that the phospholipid-dependence of some LA is related to the presence of the phospholipid-binding plasma protein β2-glycoprotein I β2-GPI) and that autoantibodies to β2-GPI might represent a specific category of LA. To verify this hypothesis we have purified IgG autoantibodies to β2-GPI from plasma of 6 patients with antiphospholipid antibody syndrome, by means of agarose-immobilized human β2-GPI. All 6 preparations tested positive in anti-β2-GPI IgG antibody ELISA and showed a marked LA activity by prolonging dilute Russell Viper Venom Time (dRVVT) from a minimum of 5.3 s in patient # 1 to a maximum of 41.1 s in patient # 3. These IgG preparations behaved as typical LA, with this activity tending to disappear in the presence of increasing phospholipid (PL) concentrations. Moreover, the LA activity of the IgG preparations was not detectable in the absence of PL, in which case the ratio between dRVVT obtained in the presence and absence of IgG autoantibodies to β2-GPI was close to 1. This pattern was confirmed by using plasma from patients with antiphospholipid antibody syndrome testing positive for anti-β2-GPI IgG antibodies. These findings suggest that dRVVT performed both in the presence and absence of PL might constitute a sensitive screening test to detect specific antibodies with LA activity.
The introduction of anticardiolipin antibody (aCL) assay, described in 1983, was able to focus much attention on the study of patients suffering from thrombosis, repeated fetal loss and thrombocytopenia, and allowing the identification of the so called antiphospholipid syndrome (APS). The identification of β2 glycoprotein I (β22GPI) as an essential component of the antigenic complex recognized by aCL suggested that this molecule could be a direct target of the antibody response. Since then, different groups have described ELISAs for the detection of anti β2GPI antibodies, applied to the clinical evaluation of patients with APS, and showing an overall better specificity. Recently, anti β2GPI were also shown to bind apoptotic bodies resulting in an alteration of their physiological clearance with the triggering of TNFα release. This observation suggests that anti β2GPI may also modify the immunogenicity of apoptotic bodies and of the autoantigens that they contain.
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