Biolayer interferometry as a novel method for detecting autoantibodies in patients with immune thrombocytopenia

Abstract

Immune thrombocytopenia (ITP) is an autoimmune hematologic disorder characterized by a low platelet count due to increased platelet destruction or decreased production. In primary ITP, the patient can have a low platelet count (<100 billion cells/L) for clinically unknown reasons. ITP is a rare disease that affects approximately 3/100 000 adults each year and some patients may experience bleeding symptoms. Autoantibody-mediated autoimmunity plays a role in the destruction of platelets by targeting platelet glycoproteins (GPs). Autoantibodies against platelet membrane GPIIbIIIa and GPIbIX are observed in about 50% of patients through direct antigen-capture assays, and 18% in patients through indirect antigen-capture assays. It is possible that some antibodies may not be detectable due to affinity or titre, or there may be other factors involved in platelet destruction. Currently, there is no definitive diagnostic test available for ITP, as a result of low assay sensitivity and different mechanisms involved in disease pathogenesis. The objective of this study was to use a novel approach to increase autoantibody detection unique to ITP patients. Total IgG was purified from patient and control plasma samples. A streptavidin-based antigen-capture assay was optimized to test the effect of biotinylation on the detection of anti-GPIIbIIIa and anti-GPIbIX autoantibodies in primary ITP patients (n=14), secondary ITP patients (n=3), non-immune thrombocytopenic controls (n=2) and healthy controls (n=16). Streptavidin-coated biosensors were used in an optimized biolayer interferometry (BLI) assay to study autoantibodies binding to biotinylated GPIIbIIIa and GPIbIX. Detection of anti-GPIIbIIIa autoantibodies in the streptavidin antigen-capture assay had a sensitivity of 24% and anti-GPIbIX autoantibodies had a sensitivity of 25%. BLI showed binding of autoantibodies in approximately 5% of ITP samples for both GPIIbIIIa and GPIbIX. The samples that had detectable autoantibodies in the antigen-capture assay did not have detectable antibodies in the BLI assay. BLI was not able to confirm antibody detection found in enzyme immunoassays.