摘要
目的:自制6A型、6B型、19A型、19F型肺炎球菌免疫血清并检测其质量,为肺炎球菌疫苗多糖含量的测定提供优质检验试剂。方法:(1)制备6A型、6B型、19A型、19F型肺炎球菌免疫血清:用6A型、6B型、19A型、19F型肺炎球菌菌株制备免疫原,按照免疫程序接种日本大耳白兔,将符合规定的免疫血清分装保存。(2)检测6A型、6B型、19A型、19F型肺炎球菌免疫血清:采用速率散射比浊法和ELISA法进行检测。通过外标法确定肺炎球菌免疫血清工作浓度,利用交叉反应对免疫血清进行特异性分析,将初步筛查出有交叉反应的免疫血清进行吸收后利用血清交叉实验进行再次筛查;使用ELISA法对自制与丹麦国家血清研究所(Statens Serum Institut,SSI)生产的肺炎球菌免疫血清的抗体滴度进行检测并对比;最后筛查出与C多糖具有交叉反应的免疫血清并进行吸收。结果:初步筛查出6A型、6B型免疫血清有交叉反应,19A型、19F型免疫血清有交叉反应,经吸收后成功去除交叉反应且反应曲线范围(多糖含量为0.75~2.0μg·mL^(-1))线性良好(r>0.985),利用ELISA法测定自制与丹麦SSI生产的肺炎球菌免疫血清的抗体滴度进行对比后发现,自制肺炎球菌免疫血清拥有良好的抗体水平;19F型血清与C多糖具有交叉反应,而6A型、6B型、19A型血清与C多糖反应的响应值与空白对照比较无明显变化;在19F型血清中添加C多糖进行吸收时,平行制备一份添加等量稀释液的19F型血清,比较吸收前后的响应值,结果一致,证明19F型抗体有效成分不会因吸收作用降低,可以按照吸收前确定的工作稀释度继续使用。结论:吸收后的自制6A型、6B型、19A型和19F型肺炎球菌免疫血清无交叉反应,抗体水平良好,可用于肺炎球菌疫苗多糖含量测定。
Objective:To prepare type 6A,6B,19A,and 19F pneumococcal immune serum and examine their qualities to provide high-quality test reagents for the determination of polysaccharide content in pneumococcal vaccine.Methods:Type 6A,6B,19A and 19F pneumococcal immune serum was prepared by using type 6A,6B,19A and 19F pneumococcal strains as immunogenes,inoculated with white rabbit according to immunization procedures,and the prescribed immune serum was aliquoted certified and stored.Then the pneumococcal immune serum was detected by rate nephelometry and ELISA.The working concentration of pneumococcal immune serum was determined by external standard method,the specificity of immune serum was analyzed by cross reaction,and the preliminarily screened immune serum was absorbed and screened again by serum cross test.ELISA method was used to detect and compare the antibody titers of self-made pneumococcal immune serum with those produced by SSI in Denmark.Finally,the immune serum with cross-reaction with C polysaccharide was screened and absorbed.Results:Cross-reaction was detected in type 6A and 6B,19A and 19F serum,the cross-reaction was successfully removed after absorption,and the reaction curve was linear in the polysaccharide content range of 0.75~2.0μg·mL^(-1)(r>0.985).ELISA method was used to determine the antibody titer of the self-made pneumococcal immune serum and that produced by SSI in Denmark,and it was found that the self-made pneumococcal immune serum had a good antibody level.Type 19F serum had cross-reaction with C polysaccharide,while the response values of type 6A,6B and 19A serum had no significant change compared with the blank control.When C polysaccharide was added to type 19F serum for absorption,type 19F serum with an equal amount of diluent was prepared in parallel,and the response values before and after absorption were compared.The results were consistent,which proved that the effective components of 19F antibody would not be reduced by absorption and could be continued to be used according to th
作者
马庆华
朱向国
花春晖
杨立蒙
郑佳
MA Qing-hua;ZHU Xiang-guo;HUA Chun-hui;YANG Li-meng;ZHENG Jia(Beijing ZhifeiLuzhu Biopharmaceutical Co.,Ltd.,Beijing 100176,China)
出处
《中国新药杂志》
CAS
CSCD
北大核心
2024年第8期818-824,共7页
Chinese Journal of New Drugs
关键词
肺炎球菌免疫血清制备
速率散射比浊法
ELISA法
多糖含量测定
pneumococcal immune serum preparation
rate scattering nephelometry
ELISA method
polysaccharide content determination
