硫酸-咔唑微孔板法检测肺炎链球菌荚膜多糖中糖醛酸含量  

Determination of uronic acid content in pneumococcal polysaccharide by sulfate-carbazole method in microplate

作　　者：任珍芸[1] 陈晓航[1] 王玺[1] 张轶[1] 张新庄[1] 刘倩[1] 刘爱萍[1] 沈荣[1] 

机构地区：[1]兰州生物制品研究所有限责任公司第一研究室,甘肃省疫苗工程技术研究中心,甘肃兰州730046

出　　处：《微生物学免疫学进展》2017年第2期36-41,共6页Progress In Microbiology and Immunology

摘　　要：目的利用微孔板检测肺炎链球菌荚膜多糖中糖醛酸含量,并对该方法进行验证及初步应用。方法以微孔板为反应容器,对常规硫酸-咔唑法的咔唑质量浓度和加热时间进行优化;并对建立的方法进行线性范围、精密度以及准确度的验证及初步应用。结果最佳的咔唑质量浓度为0.10 mg/m L,加热时间为20 min。标准品D-葡萄糖醛酸在4~40μg/m L范围内,其质量浓度与校正后吸光值呈良好的线性关系,r2>0.99;批内及批间CV值分别为1.54%~3.02%及4.53%~7.75%;加入5μg/m L、10μg/m L、20μg/m LD-葡萄糖醛酸的8-160502、9V-160102、22F-160103荚膜多糖,D-葡萄糖醛酸的回收率为92.21%~110.47%。微孔板法校正前与常规方法在测定肺炎链球菌荚膜多糖中糖醛酸含量时差异无统计学意义(P>0.05),与校正后结果差异有统计学意义(P<0.05)。微孔板法测定分子质量分布与常规方法有较好的一致性。结论硫酸-咔唑微孔板法可有效检测肺炎球菌荚膜多糖中糖醛酸含量,操作简便快捷,精密度和准确度良好,可用于肺炎链球菌荚膜多糖疫苗的质量控制。Objective To determine the uronic acid content of pneumococcal polysaccharide in the microplate , and the developed method was verified and applied. Methods Sulfate-carbazole method was conducted on the microplate, the mass concentration of carbazole and heating time were optimized and the method was set up, and to assess its linearity range, precision and accuracy, finally to make a preliminary application of the developoed method. Results The optimal mass concentration of carbazole and heating time were 0.10 mg/mL and 20 min, separately. The mass concentration of D-glucuronic acid as a standard was used in a range of 4-40 μg/mL, which showed a good linear correlation with the adjusted value of A530 , r^2 〉0.99. The CVs of determination results of pneumococcal polysaccharide in intra- and inter-assay were 1.54% -3.02% and 4.53% -7.75%, respectively. The recovery rates of D-glucuronic acid were 92.21% - 110.47%, by adding 5, 10, 20 μg/mL D-glucuronic acid to each of types 8, 9V, 22F pneumococcal polysaccharide, separately. The determined uronic acid contents in pneumococcal polysaccharide showed no a statistical meaning （P〉0. 05） between mieroplate assay not adjusted and the conventional method, but there was a statistical meaning（P〈0. 05）in determinations between microplate assay adjusted and the conventional methods. It presented a good consistency in determination of the relative molecular mass distribution in both of developed microplate assay and conventional methods. Conclusion The developed microplate assay can effectively determine uronic acid content in pneumococcal capsular polysaccharide, the method is suitable to the quality control in the process of vaccine production, with a good precision and accuracy, and easy in operation.

关 键 词：微孔板 肺炎链球菌荚膜多糖 糖醛酸 

分 类 号：R446.6[医药卫生—诊断学]