凝血酶滴定法测定宽体金线蛭抗凝血活性的影响因素

Influencing Factors in Determination of Anticoagulant Activity of Whitmania Pigra Whitman by Thrombin Titration

对测定宽体金线蛭抗凝血活性的凝血酶滴定方法的影响因素进行分析。方法：用白瓷点滴板代替试管进行滴定反应（简称白瓷板法），考察白瓷板法和常规的试管法、不同凝血酶滴定间隔时间和凝血酶浓度对抗凝血活性测定的影响。结果：在同样条件下，白瓷板法测定抗凝血活性的精确度和稳定性较试管法高。用白瓷板法测定，宽体金线蛭样品浓度在一定范围内（0．125～0．333g·ml^-1）、凝血酶浓度为20u·ml^-1或10U·ml^-1时，每隔1min滴加1次，每次5μ1，样品浓度与消耗的凝血酶体积呈线性相关，r值分别为0．961和0．992；所测得样品的抗凝血活性分别为（33．08±2．64）和（31．24±1．32）U·g^-1．RSD分别为8．0％和4．2％；对于0．333g·ml^-1的样品，其测量产生的理论误差不大于10％和5％。结论：白瓷板法可用于测定宽体金线蛭样品的抗凝血活性，用10U·ml^-1的凝血酶溶液，每分钟滴加一次，每次5山进行测定，其线性和精确度均较好。

To analyze the influencing factors in thrombin titration for the determination of anticoagulant activity of Whitmania Pigra Whitman. Methods： The white porcelain plates were used as the titration carriers instead of tubes in the titration （called white porcelain method for short）. The effect of different carriers, interval time of titration and.thromhin concentration on the results of anticoagulant activity test was studied. Results： Under the same conditions, the anticoagulant activity was more accurate and stable using white porcelain method. Using white porcelain method with 20 u·ml^-1or 10 u·ml^-1 as the thrombin concentration and titrating 5μl each time, once every minute, the thrombin consumption volume was linear with the sample concentration within the range of 0.125-0. 333 g·ml^-1 （ r20 = 0.961 and r20 = 0.992）, and the anticoagulant activity respectively was （ 33.08±2./64） and （ 31.24±1.32） u·g^-1 （RSD20= 8.0% and RSD10 = 4.2% ）. As for a certain sample concentration （0.333 g·ml^-1）, the theoretical error of determination was not more than 10% and 5%. Conclusion： The improved white porcelain method is more suitable for determining anticoagulant activity of Whitmania Pigra Whitman with more stable results and accurate end point states than tube method. Under the conditions of 10 u·ml^-1 thrombin concentration, titrating 5 μl each time, once every minute, the linearity, accuracy and precision are all promising.