速率比浊法测定13价肺炎球菌结合疫苗中的各型多糖含量

Determination of polysaccharide contents in 13-valent pneumococcal conjugated vaccine by nephelometry

目的：建立速率比浊法检测13价肺炎球菌结合疫苗各型多糖含量。方法：采用免疫化学系统（IMMAGE 800）,选择非竞争性浊度模式,样品或标准品20μL,血清20μL,反应缓冲液200μL,增益系数为4,反应时间为2.5 min;用氢氧化钠解吸附法处理样品和标准品。结果：在本文条件下,1、3、4、5、6A、6B、7F、9V、14、18C、19A、19F、23F型肺炎球菌荚膜多糖浓度在1～6μg·mL^-1检测范围内,线性良好（r〉0.99）;重复性RSD均低于7%;各型多糖含量回收率在70%～130%;专属性考察结果显示,除9N型多糖的存在会干扰9V型多糖含量检测外,13价肺炎球菌结合疫苗中不包含的多糖（2、8、10A、11A、12F、15B、17F、20、22F、33F型）对此方法没有影响;耐用性研究结果显示,氢氧化钠解吸附法处理样品和标准品时,控制在10 s左右即中和解吸附过程中加入的氢氧化钠;用所建立速率比浊法检测13价肺炎球菌结合疫苗中的1、3、4、5、6A、6B、7F、9V、14、18C、19A、19F、23F型肺炎球菌荚膜多糖含量,结果显示4批成品检测值在理论值的70%～130%之间。结论：所建立的检测方法准确,重复性好,可作为13价肺炎球菌结合疫苗的质控标准。

Objective： To establish anephelometry method for the determination of the polysaccharides contents in the 13- valent pneumococcal conjugated vaccine. Methods： Using immunochemistry system,the non- competitive nephelometric protocol was chosen,the volume of the sample or the standard substance was 20 μL,the serum volume 20 μL,the reaction buffer volume 200 μL,the gain factor 4,the reaction time 2. 5 minutes. The sample or the standard substance was treated by sodium hydroxide desorption. Results： Under these conditions in this study,linear relationships of the 1,3,4,5,6A,6B,7F,9V,14,18 C,19A,19 F,23F pneumococcal capsular polysaccharides in the range of 1- 6 μg·mL^-1 were good,r 〉0. 99. The RSD of repeatability was lower than 7%. The recoveries of 13 types of polysaccharides were 70%- 130%. The specificity results showed that 9N polysaccharide had influence on the detection of 9V polysaccharide contents,while the polysaccharides（ 2,8,10 A,11A,12 F,15B,17 F,20,22 F and33F） that were not contained in the 13- valent pneumococcal conjugated vaccine had no influence. Robustness study results showed that using sodium hydroxide to treat samples and standard substances,Na OH added during the adsorbent process could be neutralized in 10 seconds. Using the nephelometry method to determinate the contents of1,3,4,5,6A,6B,7F,9V,14,18 C,19A,19 F,23F serotype of pneumococcal capsular polysaccharides in the 13-valent pneumococcal conjugated vaccine,the results showed that in 4 batches of products the measured values were between 70%- 130% of theoretical values. Conclusion： This method is accurate and feasible. It provides a reference for the quality control of the 13- valent pneumococcal conjugated vaccine.