Abstract text (incl. figure legends and references)
Introduction:
Heparin-induced thrombocytopenia (HIT) is one of the most severe drug-reverse effect that develops in up to 5% of patients who receive heparin. Severe HIT shows a death rate of up to 20%. HIT is caused by the development of pathogenic antibodies against the complex formed between heparin and platelet factor 4 (PF4) protein. Early detection of these antibodies is crucial to prevent complications. However, current technologies such as immunoassays, and functional or quick tests for HIT diagnosis show multiple limitations such as low specificity (only ~50%).
Objectives:
As PF4 bound on cells facilitates the binding of HIT antibodies, we replace the PF4/Heparin complexes that are commonly used to capture the HIT antibodies in the standard enzyme-linked immunoassay (ELISA) by cells to develop an innovative cell-based ELISA for better detection of HIT antibodies.
Methods:
Several cell types, including breast cancer (MDA-MB-231 and MCF7), column (HCT-116), and liver (HepG2), were cultured and immobilized on the 96-well plate before adding with PF4 to capture HIT antibodies using ELISA platform. Flow cytometry and confocal microscopy were utilized to confirm the binding of HIT antibodies to cells.
Results:
Our cell-based ELISA allows us to better distinguish HIT from non-HIT antibodies as compared to the currently available ELISA for HIT diagnosis. We also identified several factors including pH, NaCl concentration, cell types and their status, and chemical fixation methods that improve the detection of HIT antibodies in cell-based ELISA.
Conclusions:
Breast cancer cell type (MDA-MB-231)-based ELISA has a strong potential to become a new material class for the development of better methods for HIT diagnosis.