Study on the bioactivity of heparin coating on the surface of centrifugal pump head by an enzyme-labeled method

Xu Suhua; Liu Jingqun; Xu Chaosheng; Huang Minju

doi:10.13498/j.cnki.chin.j.ecc.2024.05.12

Abstract：Objective To establish an enzyme-linked immunosorbent assay (ELISA) method for assessing the biological activity of heparin on the surface of centrifugal pump heads. Methods The method quantifies the biological activity of heparin by measuring the amount of thrombin inactivated on the heparinized surface saturated with antithrombin (AT). The surface of heparin binds to an excess of plasma protein AT to form an antithrombin-saturated heparinized surface (excess AT is removed through a cleaning program),which in turn binds to an excess of thrombin (Ⅱa) . The remaining thrombin (Ⅱa) reacts with the chromogenic substrate,and the peptide bond H-D-Phe Pip-Arg-pNa of thrombin is cleaved,releasing chromophores that can be tested and quantified at 405 nm. Results The kinetic absorbance at a wavelength of 405 nm on an enzyme-linked immunosorbent assay (ELISA) could be used to evaluate biological activity. Bioactivity measurements showed that the result of the control sample (uncoated centrifugal pump head) was-0.01 IU/cm2 (≤0.05 IU/cm2),and the results of all three test samples were 0.29 IU/cm2,0.40 IU/cm2,and 0.32 IU/cm2 (all≥0.1 IU/cm2),which met the validity criteria of the biological activity test. The biological activity of both the control and test samples was considered to have passed in terms of their biological activity levels. Conclusion By quantitatively testing the content of chromophores released by thrombin peptide bond cleavage using an enzyme-linked immunosorbent assay (ELISA) reader,the amount of Ⅱa reacting with heparin coating can be obtained,and the amount of AT can be further inferred to ultimately calculate the biological activity of heparin coating. This method is easy to operate,allows for simultaneous measurementof multiple samples,and has fast detection speed. The characteristics of requiring small sample size and good repeatability make it suitable for quantitative biological activity detection of heparin coatings on the surface of centrifugal pump heads.