肺炎链球菌C多糖含量检测方法的建立

Development a method in detection content for Streptococcus pneumoniae C polysaccharide

目的使用肺炎链球菌C多糖单克隆抗体(单抗),建立检测荚膜多糖中残留的C多糖含量的方法。方法选择BALB/c雌性小鼠,采用体内诱生单抗腹水,放大生产肺炎链球菌C多糖单抗;使用间接ELISA、抑制性ELISA对其进行特异性鉴定;用特异性和亲和力高的单抗尝试建立肺炎链球菌荚膜多糖中C多糖含量检测的速率比浊法,并对该方法的线性、精密度、特异性进行验证。结果所制4株单抗的抗体类别均为IgM,识别的抗原表位互不相同,亲和力也不同。间接ELISA、抑制性ELISA结果均显示,所获得的单抗能够作用于肺炎链球菌C多糖上的磷酸胆碱位点,具有很好的特异性。选择单抗E8建立速率比浊检测方法的线性、精密度和特异性均良好,检测结果表明肺炎链球菌23个血清型荚膜多糖中C多糖含量不同。结论利用单抗建立了检测肺炎链球菌荚膜多糖中残留的C多糖的含量的速率比浊法。

Objective To develop a method in detection the content of residual C polysaccharide in the capsular polysaccharide by using of monoclonal antibody (McAb) against Streptococcus pneumoniae C polysaccharide.Methods BALB/c female mice were chosen to scale up production of ascites,and each was injected of (0.5-1.0)×106 hybridoma cells.The specificity of McAbs was identified by indirect ELISA and Inhibition ELISA.The McAb with a high specificity and affinity was used in attempt to develop a rate turbidimetric method in detection C polysaccharide content in streptococcus pneumoniae capsular polysaccharide.The linearity,precision,specificity for the method were verified. Results Four McAbs belong to IgM.Their antigenic recognition epitopes and relative affinity were different.The results of the indirect ELISA and Inhibition ELISA showed that the four McAbs could specifically bind with epitopes on phosphocholine of Streptococcus pneumoniae C polysaccharide.The linearity,precision and specificity of the developed methopd were resonable.The detected result showed that the contents of C polysaccharides were different cotained in 23 serotype of Streptococcus pneumoniae capsular polysaccharides.Conclusion The detection of residual C polysaccharide content in Streptococcus pneumoniae capsular polysaccharide was developed by the rate turbidimetric method based on using the relevant McAb.