Optimization of a Western blot protocol for the detection of low levels of tissue factor in human cells.

Background: The tissue factor (TF)/activated factor VII complex is the major activator of the coagulation system. TF is expressed by a variety of cells, including activated monocytes and tumor cells. Increased TF expression can cause thrombosis in different diseases, including sepsis, viral infections, and cancer. We have previously described a method for analyzing human TF in high-expressing cells by Western blotting. Objectives: The goal of this study was to establish a method for detecting human TF in low-expressing cells. Methods: We examined the ability of 3 different antibodies to detect TF in low-expressing cell lines: rabbit polyclonal anti-human TF antibody NBP2-15139 (Novus Biologicals), goat polyclonal anti-human TF antibody AF2339 (R&D Systems), and rabbit monoclonal anti-human TF antibody ab252918 (clone EPR22548-240; Abcam). We also used the Abcam antibody to measure TF expression in lipopolysaccharide-stimulated peripheral blood mononuclear cells. Results: We found that sensitivity was affected by various factors, including the blocking conditions, the detection method, and the primary and secondary antibodies. Both the R&D and Abcam antibodies were more specific in assessing TF expression than the Novus antibody; however, the Abcam antibody was the best of the 3 in evaluating TF in low-expressing cell lines. We detected TF in lipopolysaccharide-stimulated human peripheral blood mononuclear cells using the new method with the Abcam antibody. Conclusion: Researchers should consider each step in Western blotting when establishing a method for detecting low-abundance antigens, such as TF.