b型流感嗜血杆菌多糖竞争ELISA检测方法的建立

Competitive ELISA Method for Determining Haemophilus Influenza Type b Polysaccharide

目的建立竞争酶联免疫吸附分析法(Enzyme-Linked Immunosorbent Assay,ELISA),测定b型流感嗜血杆菌多糖(PRP)浓度方法以b型流感嗜血杆菌多糖-酪胺(Ty)为包被抗原,待测多糖为竞争抗原,与优化的抗b型流感嗜血杆菌多糖抗体反应,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b型流感嗜血杆菌多糖-酪胺包被浓度为1∶400倍稀释,抗多糖血清稀释度为1:40K。检测线性范围6.25μg/ml～100μg/ml,最低检测限为3.13μg/ml,回归方程为B/B0=-34.328[PRP]+105.03,线性相关系数为R2=0.995。批内精密度为3.4%～6.5%,批间精密度为8.48%,测定培养基中b型流感嗜血杆菌多糖的回收率为95.7%。结论本研究建立的b型流感嗜血杆菌多糖竞争ELISA方法准确性和精密性均较好,可以特异性地检测b型流感嗜血杆菌多糖浓度。

Objective To develop competitive ELISA method for determining the concentration of haemophilus in- fluenza type b polysaccharide （PRP）. Method PRP - Ty was set as coating antigen and poliasccharide as com- petition antigen, comparison were made between the two reactions against antisera of haemophilus influenza type b. The accuracy and precision of the method were validated accordingly. Results The optimal coating con- ccntration of PRP - Ty and serum dilution factor was 1 ： 400, and 1 ： 40K respectively. Quantitation of PRP concentration was linear from 6. 25ug/ml - 100ug/ml, with lower limit of detection being 3. 13ug/ml. The re- gression equation was B/Bo = -34. 328 [PRP] ＋ 105.03 with RSQ =0. 995. The coefficient of variation of intra - assay and inter - assay was 3.4% -6. 5% and 8.48% respectively. The recovery rate of PRP in culture medium was 95.7%. Conclusion The competitive ELISA method showed both high accuracy and precision, and thus can be used for detection of haemophilus influenza type b polysaccharide.