PB1102 - Evaluation of the ability of commercial ELISAs to measure tissue factor in human plasma

Background: Tissue factor (TF) -positive extracellular vesicles (EVs) are released into the circulation in various diseases. Antigen-based assays, such as enzyme-linked immunosorbent assays (ELISAs), are easy, fast and commonly used to measure proteins in plasma, including TF. However, there are several challenges with measuring proteins in plasma.

Aims: We evaluated the ability of four commercial ELISAs to measure human TF in plasma.

Methods: We determined the ability of 4 commercial ELISAs (Imubind, Quantikine, Human SimpleStep, and CD142 Human) to measure recombinant human TF (Innovin), TF-positive EVs isolated from a human pancreatic cancer cell line (HPAF-II), TF-positive EVs isolated from plasma from LPS-stimulated whole blood (n=5) and TF-positive EVs isolated from acute leukemia patient plasma (n=6). We measured TF activity of isolated EVs using a factor Xa generation assay. Levels of TF activity in the EVs in the from the cell line, acute leukemia patients and LPS stimulated blood were designated high, high and medium, respectively.

Results: Imubind, Quantikine and Human SimpleStep detected recombinant TF in a concentration-dependent manner. In contrast, the CD142 Human ELISA failed to detect recombinant TF. Imubind and Quantikine detected TF in EVs isolated from cell culture supernatants from a human pancreatic cancer cell line (high TF). Imubind and Quantikine also detected TF on EVs isolated from plasma from acute leukemia patients with high TF activity levels and Quantikine detected a TF-like signal in the EV-free plasma (high TF). Importantly, none of the ELISAs detected TF antigen in plasma or EVs isolated from LPS-stimulated whole blood (medium TF).

Conclusion(s): Commercial ELISAs have low sensitivity and specificity to measure TF in human plasma. This appears to be due to the high background of plasma samples, particularly with Imubind. We do not recommend using these ELISAs to measure TF in human plasma.