酶标板法检测甘油三酯净含量方法的优化、验证及其在人血白蛋白层析纯化工艺中的应用

Optimization and validation of enzymatic detection for net content of triglyceride and its application in chromatography purification process of human serum albumin

目的优化酶标板法检测甘油三酯（triglyceride,TG）净含量的方法,进行验证,并将其应用于监测人血白蛋白层析纯化工艺中样品的脂类含量,对工艺优化提供指导。方法建立酶标板上快速检测TG含量方法,通过两步酶反应的吸光度值的差值计算TG净含量。确立该方法的标准曲线范围,并进行准确度和精密度进行验证。采用该方法监测层析工艺中样品的TG净含量,优化人血白蛋白层析纯化工艺。结果该方法检测TG的线性范围在78-2500μg/ml之间,游离甘油（free glycerin,FG）的线性范围在8.13-260μg/ml之间,R2均为0.997。各样品检测结果 CV均〈4%,回收率在102%-107%之间,准确度较好;同一样品由同一个实验员在同一试验中连续测定6次和由不同的实验员在不同日期内分别测定3次的结果,CV均〈3%,精密度较好。在人血白蛋白层析纯化工艺中采用去脂剂可将白蛋白样品中的残余脂类去除。结论优化后的甘油三酯净含量检测方法为人血白蛋白层析纯化工艺的优化提供了指导,在血液制品工艺研发过程中具有重要的应用价值。

Objective To optimize and validate the enzymatic detection of net content of triglyceride（TG）and apply to monitoring the lipid content of samples in chromatography purification process of human serum albumin（HSA）. Methods A method for rapid detection of TG content on micro-titer plate by calculating TG levels from the difference between the absorbance values of the two-step enzymatic reaction. The method was determined for linear range of standard curve,and validated for accuracy and precision. The net content of TG of samples in chromatography process was monitored by the method,based on which the chromatography purification process of HSA was optimized. Results The linear range of standard curve for detection of TG was 78 - 2 500 μg / ml,while that for detection of free glycerin（FG） was 8. 13 -260 μg / ml,both with R2 values of 0. 997. All the CVs of detection results of samples were less than 4%,while the recovery rate was 102% - 107%,indicating a high accuracy. The identical sample was detected for 6 times by one personnel in one test and for 3 times by various personnel on various dates,of which the CVs of results were less than 3%,indicating a high precision. The lipid residue in the HSA samples was removed by application of degreasing agent in chromatography purification process. Conclusion The detection of net content of TG after optimization provided a guidance for the optimization of chromatography purification process of HSA,which was of an important significance in the development of preparation process of blood products.