b型流感嗜血杆菌多糖竞争ELISA检测方法的建立与应用

Establishment and application of competitive ELISA method for detertion of Haemophilus influenzae type b polysaccharide

目的建立检测b型流感嗜血杆菌（Haemophilus in fluenzae typeb，Hib）多聚核糖基核糖醇磷酸盐（polyrmosylribitol phosphate，PRP）的竞争酶联免疫吸附法（competative enzyme-linked immunosorbent assay，cELIsA）。方法以HibPRP-酪胺（PRP-Ty）为包被抗原，待测PRP为竞争抗原，利用方阵滴定法确定抗原与血清抗PRP抗体的最适反应条件，建立cELISA法并验证其准确性和精密度，并将该法与传统检测法进行比较。结果最适包被PRP-Ty浓度为1．30mg／L，血清抗PRP抗体稀释度为1：40000。该法的检测线性范围为6．25-100．00mg／L，决定系数（R^2）为0．99，批内精密度为3．39％～6．53％，批间精密度为8．48％。该法对HibPRP的检测结果与传统化学法一致。结论建立的Hib多糖cELISA法的准确性和精密性良好，可特异性检测HibPRP。

Objective To establish competitive ELISA （cELISA） method for determining polyri- bosylribitol phosphate （PRP） of Haemophilus influenzae type b （Hib）. Methods PRP-Ty was used as coating antigen and Hib PRP sample was used as competitive antigen. Optimal reaction conditions were determined between antigens and serum antibodies to Hib PRP by criss-cross serial dilution analysis. The cELISA method was established and its accuracy and precision were validated. The cELISA method was compared with the traditional method. Results Optimal coating concentration of PRP-Ty and antiserum dilution were 1.30 mg/L and 1 ： 40 000, respectively. The linear range of the cELISA method was from 6.25 mg/L to 100.00 mg/L, and the coefficient of determination （R2 ） was 0.99. The precision of intra- assay and inter-assay were 3.39%-6.53% and 8.48%, respectively. The detection results of Hib PRP with the cELISA method were consistent with those with the traditional chemical method. Conclusion The cELISA method has better accuracy and precision, and can be used for detection of Hib polysaccharide.