人凝血/抗凝血因子类产品蛋白含量快速检测方法的建立及验证

Development and verification of a method for rapid determination of protein content in human coagulation / anticoagulation factor products

目的建立人凝血因子Ⅷ、人凝血因子Ⅸ、人凝血酶、人抗凝血酶Ⅲ蛋白含量Lowry快速检测法,并进行验证。方法采用《中国药典》三部(2010版)Lowry法检测因子类产品的蛋白含量,通过耐用性研究降低辅料对方法的干扰,优化Lowry检测方法,并对方法的专属性、线性及范围、准确度、精密度进行验证。结果不同辅料对Lowry法的干扰程度不同,人凝血因子Ⅷ、人凝血因子Ⅸ、人凝血酶、人抗凝血酶Ⅲ样品分别稀释20、10、80、160倍,可明显降低辅料的影响;Lowry法与凯氏定氮法相关性良好,方法线性R>0.99,其准确度、精密度均良好。结论 Lowry法测定人凝血因子Ⅷ、人凝血因子Ⅸ、人凝血酶、人抗凝血酶Ⅲ蛋白含量操作简便、快速、准确,可用于产品的质量控制。

Objective To develop and verify a Lowry method for rapid determination of protein contents in human coagulation factor Ⅷ（FⅧ）, factor Ⅸ（FⅨ）, human thrombin and anti-thrombin Ⅲ（AT Ⅲ）. Methods The protein contents in above-mentioned products were determined by the Lowry method in Chinese Pharmacopoeia（Volume Ⅲ, 2010edition）. The method was optimized by decreasing the interference of auxiliary materials by robustness test, and verified for specificity, linearity and range of standard curve, accuracy and precision. Results The interference levels of various auxiliary materials to Lowry method were different, which decreased significantly by dilution of F Ⅷ, FⅨ, human thrombin and AT Ⅲ by 20, 10, 80 and 160 folds respectively. The determination result by Lowry method showed good correlation with that by Kjeldahl method, with a R value of more than 0. 99. Lowry method showed high accuracy and precision.Conclusion Lowry method was simple, rapid and accurate for determination of protein contents in Fr Ⅷ, F Ⅸ, human thrombin and ATⅢ, which might be used for the quality control of the products.