Journal: Research and Practice in Thrombosis and Haemostasis

Article Title: A novel factor IXa–specific enzyme-linked immunosorbent assay detects factor IXa in human plasma

doi: 10.1016/j.rpth.2024.102338

Figure Lengend Snippet: Western blot analysis of purified proteins under reducing conditions. (A) Monoclonal antibody 16E11, raised against a peptide with the sequence of the new N-terminus that is generated when factor (F)IX is cleaved after R226, detected the heavy chains (HC) of FIXaβ and FIXaα and activated FIX (FIXa) complexed to antithrombin (AT) (FIXa-AT), but did not detect FIXα. (B) Monoclonal antibody 13B10, raised against the N-terminus of FIX, detected FIX and the light chains (LC) of FIXaβ, FIXaα, and FIXα. Cleavage of FIX to FIXaα or FIXα was not quantitative; thus, the parental FIX remained in these preparations . Sodium dodecyl-sulfate polyacrylamide gel electrophoresis and Western blotting were performed as described in Methods.

Article Snippet: 13B10 also binds to mouse FIX (Sino Biological) and mouse FIXa but with lower affinity than for the corresponding human proteins ( B).

Techniques: Western Blot, Purification, Sequencing, Generated, Polyacrylamide Gel Electrophoresis

Journal: Research and Practice in Thrombosis and Haemostasis

Article Title: A novel factor IXa–specific enzyme-linked immunosorbent assay detects factor IXa in human plasma

doi: 10.1016/j.rpth.2024.102338

Figure Lengend Snippet: Calcium dependence of monoclonal antibodies 16E11 and 13B10. (A) Monoclonal 16E11 bound factor (F)IXa equally ± 5mM CaCl 2 but did not bind FIX. (B) Monoclonal 13B10 bound both FIXa and FIX optimally in the absence of CaCl 2 . Addition of 5 mM CaCl 2 dramatically decreased binding. Binding was determined as described in Methods. The graphs are representative curves.

Article Snippet: 13B10 also binds to mouse FIX (Sino Biological) and mouse FIXa but with lower affinity than for the corresponding human proteins ( B).

Techniques: Binding Assay

Journal: Research and Practice in Thrombosis and Haemostasis

Article Title: A novel factor IXa–specific enzyme-linked immunosorbent assay detects factor IXa in human plasma

doi: 10.1016/j.rpth.2024.102338

Figure Lengend Snippet: Factor (F)IXa enzyme-linked immunosorbent assay (ELISA) titrations and plasma activation. The specificity of the FIXa ELISA for various FIX forms was examined using purified proteins. (A) The FIXa-specific ELISA detected both free FIXaβ and FIXaβ-antithrombin (AT) complexes but not FIX. (B) The FIXa-specific ELISA detected FIXaβ and FIXaα, but not FIXα. As explained in the Results, the FIXaα preparation contained approximately 40% unactivated FIX (by Western blot analysis), so a 60% FIXaβ/40% FIX mixture was included to reflect how an approximately equivalent number of FIXa heavy chains within the FIXaα population would react in the assay. (C) The FIXa-specific ELISA detected the time-dependent generation of FIXa in human plasma. Citrated pooled normal plasma (PNP) or FIX immune-depleted (ID) plasma was recalcified and activated with either tissue factor (TF) or FXIa as described in Methods. The FIXa ELISA was used to measure the increase in plasma FIXa in activated PNP over time. FIX ID plasma did not have measurable FIXa, even after activation. (D) Addition of heparin (Hep) resulted in an increase in FIXa-AT. The FIXa-specific ELISA or the FIXa-AT ELISA was used to measure the increase in plasma FIXa in PNP after activation with FXIa for 3 minutes at 37 °C. Hep was added after quenching, as indicated (light blue). The graphs are representative curves.

Article Snippet: 13B10 also binds to mouse FIX (Sino Biological) and mouse FIXa but with lower affinity than for the corresponding human proteins ( B).

Techniques: Enzyme-linked Immunosorbent Assay, Activation Assay, Purification, Western Blot