·Immunization of mice, hybridoma technology, cell expression and purification were used to prepare the anti-FⅨa monoclonal antibodies of high purity. The monoclonal antibodies with high affinity for FⅨa were screened by enzyme-linked immunosorbent assay. The activated partial thromboplastin time (APTT) and prothrombin time (PT) were used to evaluate the antithrombotic effects of the monoclonal antibody. And then chromogenic substrate method was used to detect the effect of monoclonal antibody on the enzyme activity of FⅨa. The method of computer simulation of protein-protein docking was adopted to predict the possible binding sites between FⅨa and the antibody, and the binding site, was verified by competitive experiments (indirectly through the chromatic substrate method).

·FⅨa-4, an anti-FⅨa monoclonal antibody with high affinity, was generated. Although FⅨa-4 did not affect PT and the enzyme activity of FⅨa, it significantly prolonged APTT to 88.8 s, which was 3.5 times of the control group (25.5 s), in a concentration-gradient dependence. The protein-protein docking prediction results revealed that FⅨa-4 did not directly bind to substrate catalytic sites of FⅨa, but occupied the binding region of FⅨa and FⅧa. Competitive experiments further verified the above results. FⅨa-4 inhibited FⅩa production in a dose-dependent manner, almost completely inhibiting FⅩa production at the concentrations of 400 pmol/L, and FⅧa could correct the inhibition effect of the antibody up to nearly 50%.

·The monoclonal antibody against FⅨa-4 is obtained. FⅨa-4 competes with FⅧa to bind FⅨa, and inhibits the conversion of FⅩ to FⅩa which is catalyzed by FⅧa-FⅨa complex; it plays an antithrombotic role mainly by inhibiting endogenous coagulation pathway.