论文部分内容阅读
目的微孔板法检测肺炎球菌多糖中甲基戊糖含量,并对该方法进行验证。方法将常规甲基戊糖测定法应用于微孔板,并优化盐酸半胱氨酸浓度。对建立的方法进行线性、精密度及准确度的验证。用建立的方法检测肺炎球菌多糖样品中甲基戊糖含量及多糖衍生物的分子量分布。结果最佳的盐酸半胱氨酸浓度为15 mg/ml。标准品L-鼠李糖在5~30μg/ml范围内,其浓度与(A396-A430)值呈良好的线性关系,R2>0.999;批内及批间CV值分别为0.9%~1.1%及2.2%~3.2%;加入2.5、5、10μg/ml L-鼠李糖的6B型原糖及多糖衍生物中的甲基戊糖含量的回收率为91.73%~100.70%。微孔板法及常规方法测定的肺炎球菌多糖中甲基戊糖含量无明显差异(P>0.05),微孔板法测定多糖分子量分布与常规方法有较好的一致性。结论微孔板法可有效、准确、稳定地检测肺炎荚膜多糖及其衍生物中甲基戊糖含量,该方法灵敏度高,操作简便快捷,节约样品、试剂等材料,适用于疫苗生产过程中的质量控制。
Objective To detect the content of methyl pentose in pneumococcal polysaccharide by microplate method and verify the method. Methods The conventional methyl pentose assay was applied to microplates and the cysteine hydrochloride concentration was optimized. The established method of linear, precision and accuracy of the verification. The established method was used to detect the content of methyl pentose in the samples of pneumococcal polysaccharide and the molecular weight distribution of polysaccharide derivatives. Results The best cysteine hydrochloride concentration was 15 mg / ml. The standard L-rhamnose was in the range of 5 ~ 30μg / ml. The concentration of L-rhamnose showed a good linear relationship with (A396-A430), R2> 0.999. The intra- and inter-assay CV values were 0.9% -1.1% 2.2% ~ 3.2%. The recoveries of methyl pentose content in crude extracts of type 6B and polysaccharide with addition of 2.5, 5, 10μg / ml L-rhamnose were 91.73% ~ 100.70%. Microplate method and conventional method for determination of pneumococcal polysaccharide methyl pentose content was no significant difference (P> 0.05), microplate method polysaccharide molecular weight distribution and conventional methods have good agreement. Conclusion Microplate method can effectively, accurately and stably detect the content of methyl pentose in pneumonia capsular polysaccharide and its derivatives. The method has the advantages of high sensitivity, simple and quick operation, saving samples, reagents and other materials and is suitable for the production of vaccines Quality control.