CWPS竞争ELISA检测方法的建立

Development of competitative ELISA for quantitation of pneumococcal cell wall polysaccarides

目的：建立竞争酶联免疫吸附法（enzyme-linked immunosorbent assay，ELISA）测定C-多糖（Cell Wall Polysaccharides，cwes）浓度的方法。方法：以C-多糖为包被抗原，待测多糖为竞争抗原，与优化的抗C-多糖抗体反应，建立标准曲线，并验证该方法的准确性和精密度。结果：优化后的C-多糖包被浓度为10μg·mL^-1，抗C-多糖血清稀释度为1：160000。检测线性范围1250～20 ng·mL^-1，最低检测限为10ng·mL^-1，回归方程为B／B0=-0.2521 lg[CWPS]＋0.9529，线性相关系数为R^2=0.99。批内和批间精密度分别为9.7％和6.9％，测定PS6A精糖中标准CWPS的回收率为100.3％。结论：本研究建立的竞争ELISA方法，准确性和精密度均较好，可以检测肺炎链球菌荚膜多糖中CWPS的浓度。

Objective： To develop competitive ELISA for determination of pneumococcal cell wall polysaccharides （CWPS） concentration. Methods： The standard curve was established by using CWPS as coating antigen, competing with polysaccharide in samples to react against antisera of pneumococcal ce The accuracy and precision of the method were validated accordingly. Results： The results 11 wall polysaccharides. showed that the optimal coating concentration of CWPS and serum dilution factor was 10 μg·mL^-1, and 1：160 000 respectively. The relation between CWPS concentrations and their reaction was linear in the range of 20～1 250 ng· mL^-1 CWPS concentration with the lowest limit of detection being 10 ng· mL^-1. The regression equation was B/B0-0.252 1 lg [CWPS] ＋ 0.952 9 with RSQ = 0.99. The coefficient of variation of intra-assay and inter-assay was lower than 9.7% and 6.9%, respectively. The recovery rate of CWPS in pneumococcal polysaccharide serotype 6A was 100.3%. Conclusion： The competitive ELISA method developed showed high accuracy and precision. The method can be used for detection of CWPS in pneumococcal polysaccharide