Abstract:

In order to obtain plasmin-α2-plasmin inhibitor complex (PIC) and thrombin-antithrombin (TAT) monoclonal antibodies and establish a detection method of the corresponding complex, it is necessary to prepare high-purity PIC and TAT complexes. With lipemia plasma as the research material, the plasma pretreatment was first performed. Plasma was activated by urokinase and passed through Lysine-sepharose affinity column to finally obtain high-purity PIC. After the treated plasma is adsorbed by barium chloride and precipitated with saturated ammonium sulfate, antithrombin and prothrombin are obtained. Ecarin snake venom is used to activate prothrombin to obtain thrombin. The thrombin and antithrombin were passed through Heparin-sepharose, respectively. Affinity column was used to improve its purity, and it was mixed and reacted in vitro to obtain a TAT mixture, and then through a Heparin-sepharose affinity column to remove unreacted thrombin and antithrombin, and finally obtain high-purity TAT. The product was identified and quantified by SDS-PAGE, ultraviolet spectrophotometry and HISCL automatic analyzer. The results showed that the best activation condition for PIC was 2 mg urokinase/100 mL plasma, activated at 37 ℃ for 30 min; 2. 5 mg PIC was obtained, with a purity of more than 90% identified by SDS-PAGE. The activation condition of prothrombin was 0. 5 mL snake venom/100 mL plasma, activated at

37 ℃ for 20 min, and finally 2. 1 mg thrombin and 6. 2 mg antithrombin were obtained from 100 mL plasma. The optimal reaction mass ratio of thrombin and antithrombin in vitro was 0. 7 ∶ 1, and the purity of TAT was more than 80% identified by SDS-PAGE.

Key words: thrombin, antithrombin, plasmin-α2-plasmin inhibition, affinity chromatography, purification