A QCM-Based Biosensor to Detect HIT-like Antibodies: Differentiating KKO from RTO via FcγRIIA Interactions

Abstract

Heparin-induced thrombocytopenia (HIT) is a serious side effect that occurs in patients undergoing heparin therapy. The known risk factor is the presence of antibodies created against platelet factor 4 and heparin complexes (PF4/heparin) in the blood, which activate platelet Fc receptors (FcγRIIA). Although immunoassays have been developed for HIT diagnosis, their specificity remains low (∼50%) due to the binding of nonpathogenic antibodies to the same antigen (PF4/heparin). As a result, a lack of rapid, highly sensitive, and selective diagnostic tests poses challenges for HIT treatment. In this study, we used monoclonal HIT-like KKO antibodies as a model and demonstrated a rapid biosensor based on quartz crystal microbalance (QCM) that effectively distinguishes the pathogenic (KKO) from nonpathogenic (RTO) antibodies within 10 min. Based on our key finding, KKO and RTO present clear binding affinity differences against FcγRIIA: RTO binds to FcγRIIA while KKO can bind to FcγRIIA only in the presence of PF4/heparin. The determined negative zeta potentials of proteins confirmed that the observed affinity differences toward FcγRIIA result from specific binding rather than nonselective electrostatic interactions. This highlights the potential use of FcγRIIA as an antigen instead of traditional PF4/heparin complexes. Although only a monoclonal HIT-like antibody was tested, the use of FcγRIIA to distinguish the binding patterns of KKO and RTO could be extended to the detection of human HIT antibodies.