摘要
目的优化检测人凝血因子Ⅷ(FⅧ)蛋白质含量的BCA法,并进行验证。方法对BCA法检测人FⅧ的蛋白质含量进行优化:模拟试管法进行化学反应,微量板法进行读数和计算;对优化后的BCA法的耐用性、标准曲线的线性和范围、准确性、重复性、中间精密性进行验证,并与传统的钨酸沉淀法、免疫散射比浊法和优化的凯氏定氮法的检测结果进行比较。结果 BCA法检测人血白蛋白(human serum albumin,HSA)蛋白质含量的可信区间为100～800μg/ml。甘氨酸和赖氨酸的浓度小于125 mmol/L时,对BCA法检测结果无干扰;以HSA作为标准品绘制的标准曲线的可信区间为62.5～1 000μg/ml,人FⅧ工艺样品在以HAS和BSA作为标准品绘制的两种标准曲线下的检测值差异无统计学意义(P>0.05);BCA法的准确性、重复性、中间精密性均符合验证要求;BCA法检测结果与优化后的凯氏定氮法相关性良好。结论优化了检测人FⅧ蛋白质含量的BCA方法,该法用于人FⅧ生产工艺中间品的质量控制是可行的。
Objective To optimize and verify the BCA method for determination of protein content in human coagulation factor Ⅷ（FⅧ ）. Methods The BCA method for determination of protein content in FⅧ was optimized by combination of tube assay in chemical reaction with micro-plate assay in data calculation. The optimized BCA method was verified for ro- bustness, linearity and range of standard curve, accuracy, reproducibility and intermediate precision, and the results were compared with those of traditional tungstic acid precipitation, immune scatter turbidimetry and modified Kjeldahl method. Results The confidence interval of protein content in human serum albumin （HSA） determined by BCA method was 100 ～800 μg/ml. Glycine and lysine at concentrations of less than 125 mmol/L showed no interference to the de- termination result by BCA method. The confidence interval of standard curve plotted using HSA as a standard was 62. 5 ～ 1 000 μg / ml. The determination results of test samples of FVⅧ by standard curves using HSA and BSA as standards showed no significant difference（P 〉 0. 05）. The accuracy, reproducibility and intermediate precision of BCA method met the requirements for verification. The determination result by BCA method showed good correlation with that by modified Kjeldahl method. Conclusion The BCA method for determination of protein content in human FⅧ was optimized, which was feasible to be used for quality control of intermediates during production of human FⅧ.
出处
《中国生物制品学杂志》
CAS
CSCD
2013年第10期1488-1492,共5页
Chinese Journal of Biologicals
