细菌内毒素对A群C群脑膜炎奈瑟菌荚膜多糖蛋白质含量测定结果的影响
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摘要
目的探究Lowry法测定A群C群脑膜炎奈瑟菌(简称脑膜炎球菌)多糖原液蛋白质含量时,细菌内毒素对Lowry法的干扰情况。方法根据《中华人民共和国药典》2015版(三部)规定的方法,测定含不同浓度的细菌内毒素的A群C群脑膜炎球菌多糖原液的蛋白质含量,对检测结果进行分析。以细菌内毒素为干扰物质,设计3种方法进行验证,对验证结果进行离散系数,即CV值分析,确定干扰限值。结果当细菌内毒素含量较高时,A群C群脑膜炎球菌多糖原液的蛋白质含量明显增高,该结果可能为假阳性。验证试验结果显示,当溶液中细菌内毒素浓度达到0.313 EU/mL时,会对检测结果产生干扰,浓度越高,干扰程度越高。结论为获得准确的检测结果,当细菌内毒素浓度达到干扰限值时,应先消除干扰,再测定蛋白质含量。
Objective To investigate the interference from bacterial endotoxin on the Lowry method when this method was used in determination the protein content contained in group A/C meningococcal polysaccharides. Methods The protein content was determined in group A/C meningococcal polysaccharides solution with different concentrations of bacterial endotoxin according to the method published in the 《Chinese Pharmacopoeia》(2015 edition), and the detection results were analyzed. Three methods were designed in verification, by using bacterial endotoxin as an interfering substance, and the verified result was subjected to a coefficient of variation(CV), that is, in analysis of CV value to determine the interference limit. Results As the bacterial endotoxin content was high, the protein content in group A/C meningococcal polysaccharides was significantly increased, and the result may be a false positive. The verification test results showed that the bacterial endotoxin content in the solution reached to 0.313 EU/mL, the detection result will be disturbed. The high level of the bacterial endotoxin content resulted in the high interference degree. Conclusion In the case of the bacterial endotoxin content reaches to the interference limit, the interference should be eliminated first, and protein content determination performed afterward, in order to obtain accurate test results.
Objective To investigate the interference from bacterial endotoxin on the Lowry method when this method was used in determination the protein content contained in group A/C meningococcal polysaccharides. Methods The protein content was determined in group A/C meningococcal polysaccharides solution with different concentrations of bacterial endotoxin according to the method published in the 《Chinese Pharmacopoeia》(2015 edition), and the detection results were analyzed. Three methods were designed in verification, by using bacterial endotoxin as an interfering substance, and the verified result was subjected to a coefficient of variation(CV), that is, in analysis of CV value to determine the interference limit. Results As the bacterial endotoxin content was high, the protein content in group A/C meningococcal polysaccharides was significantly increased, and the result may be a false positive. The verification test results showed that the bacterial endotoxin content in the solution reached to 0.313 EU/mL, the detection result will be disturbed. The high level of the bacterial endotoxin content resulted in the high interference degree. Conclusion In the case of the bacterial endotoxin content reaches to the interference limit, the interference should be eliminated first, and protein content determination performed afterward, in order to obtain accurate test results.
引文
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[7] GONG X W,WEI D Z H,HE M L.Lowry method for the determination of pegylated proteins:the error,its reason,and a method for eliminating it[J].Anal Biochem,2006,354(1):157-158.
[8] 郑丽钦.探讨甘氨酸、甘露醇对Lowry法测蛋白含量的干扰[J].海峡药学,2006,18(4):81-83.
[9] 路苹,于同泉,王淑英,等.蛋白质测定方法评价[J].北京农学院学报,2006,21(2):65-69.
[10] DOGAR V,SOKHEY J,KUMAR S,et al.Interference of thiomersal in biologicals during protein estimation[J].Biologicals,2002,30(4):271-275.
[11] 菅原洁,副岛正美.蛋白质的定量法[M].张旭,译.2版.北京:农业出版社,1981:133.
[12] BRUSCALUPI G,LEONI S,PANZALI A F,et al.Interference of some detergent micelles with the titration of protein[J].Boll Soc Ital Biol Sper,1979,55(5):450-454.
[13] HERD J K.Interference of hexosamines in the Lowry reaction[J].Anal Biochem,1971,44(2):404-410.
[2] 国家药典委员会.中华人民共和国药典(三部)[S].北京:中国医药科技出版社,2015:71-73.
[3] LOWRY O H,ROSEBROUGH N J,FARR A L.Protein measurement witn the folin phenol reagent[J].J Biol Chem,1951,193:265-275.
[4] 贺建华,邵纯君,鹿麟,等.福林酚法测定甘露聚糖肽口服溶液中蛋白质的含量[J].中国药物评价,2017,34(2):92-94.
[5] 卫辰,骆鹏,王丽婵,等.两种Lowry 法检测百日咳疫苗原液蛋白含量的比较[J].中国生物制品学杂志,2017,30(5):519-524.
[6] 李新国,袁军,张洋,等.Lowry法测定b型流感嗜血杆菌结合疫苗蛋白含量干扰现象的排除[J].中国生物制品学杂志,2009,22(10):1029-1031.
[7] GONG X W,WEI D Z H,HE M L.Lowry method for the determination of pegylated proteins:the error,its reason,and a method for eliminating it[J].Anal Biochem,2006,354(1):157-158.
[8] 郑丽钦.探讨甘氨酸、甘露醇对Lowry法测蛋白含量的干扰[J].海峡药学,2006,18(4):81-83.
[9] 路苹,于同泉,王淑英,等.蛋白质测定方法评价[J].北京农学院学报,2006,21(2):65-69.
[10] DOGAR V,SOKHEY J,KUMAR S,et al.Interference of thiomersal in biologicals during protein estimation[J].Biologicals,2002,30(4):271-275.
[11] 菅原洁,副岛正美.蛋白质的定量法[M].张旭,译.2版.北京:农业出版社,1981:133.
[12] BRUSCALUPI G,LEONI S,PANZALI A F,et al.Interference of some detergent micelles with the titration of protein[J].Boll Soc Ital Biol Sper,1979,55(5):450-454.
[13] HERD J K.Interference of hexosamines in the Lowry reaction[J].Anal Biochem,1971,44(2):404-410.