An ELISA system to detect anti-factor VIII antibodies without interference by lupus anticoagulants. Preliminary data in hemophilia A patients

Abstract

BACKGROUND AND OBJECTIVES. Difficulties in identifying the coexistence of neutralizing anti-factor VIII antibodies (anti-fVIII) and lupus anticoagulant (LA) are mainly due to the interference of LA on anti-fVIII assays. Our aim was to reveal the presence of anti-fVIII using a system that is not affected by LA. DESIGN AND METHODS. We developed an enzyme-linked immunosorbent assay (ELISA) method that uses phospholipid-free recombinant factor VIII as the antigen. A monoclonal anti-fVIII was tested as a positive control, excluding non-specific binding by using two unrelated monoclonal antibodies. The ELISA was performed on hemophilic plasmas with anti-fVIII and negative LA (n=12) or without inhibitors (n=12). Two hemophilic plasmas with LA and presumably anti-fVIII were also assayed. Positive LA (n=12) and normal (n=10) plasmas were tested as negative controls. RESULTS. All (12/12) plasmas with anti-fVIII and 5/12 hemophilic plasmas without inhibitors were positive; LA and normal plasma controls were negative. INTERPRETATION AND CONCLUSIONS. Results presented here show that LA does not interfere with the anti-fVIII ELISA. However, the assay detects both neutralizing and non-neutralizing anti-fVIII antibodies, therefore a neutralizing effect must be confirmed through functional tests.