摘要目的 建立检测19A型肺炎链球菌荚膜多糖的竞争ELISA方法,检测细菌发酵过程中多糖的表达量.方法 以19A型肺炎链球菌多糖为包被物,待测多糖为竞争物,建立间接竞争ELISA方法并验证该方法的准确性和精密度.用建立的方法检测不同培养条件下发酵物中的多糖表达量.结果 最佳多糖包被质量浓度为10 mg/L,多糖抗血清稀释度为1∶4×104.当检测范围为39.06～5 000.00μg/L时,决定系数为0.995.批内和批间相对标准差分别为5.08％～9.58％和5.28％～12.93％.培养物样品加标回收率为95.84％～110.07％.两种不同培养条件下培养物中的多糖表达量分别为312.17 mg/L和1 056.42 mg/L.结论 新建的方法能用于19A型肺炎链球菌发酵物中多糖表达量的检测,对于优化培养条件及掌握培养物收获时间具有指导意义.

abstractsObjective To develop a competitive ELISA method for quantitative determination of pneumococcal polysaccharide serotype 19A (PS19A) in fermentation liquid.Methods PS19A and test polysaccharide were used as coating and competitive antigens,respectively,to establish a competitive ELISA method.The accuracy and precision of the method were validated.The expression of polysaccharide under two different culture conditions was determined using this method.Results The optimal coating concentration of PS19A and serum dilution were 10 mg/L and 1 ∶ 4 × 104,respectively.The linear range was 39.06-5 000.00 μg/L and the coefficient of determination was 0.995.The relative standard deviations of intra-assay and inter-assay were 5.08％-9.58％ and 5.28％-12.93％,respectively.The recovery of standard addition was 95.84％-110.07％.The expression of polysaccharide under two culture conditions was 312.17 and 1 056.42 mg/L.Conclusion The method can be used for detection of PS19A expression in cultures,which provides a reference for optimization of culture conditions.