b型流感嗜血杆菌多糖间接竞争ELISA检测方法的建立

Development of an indirect competitive ELISA method for determination of Haemophilus influenzae type b polysaccharide

目的建立b型流感嗜血杆菌（Haemophilus influenzae type b,Hib）多糖间接竞争ELISA检测方法,并进行验证及初步应用。方法以Hib多糖—酪胺结合物（PRP-Ty）作为包被抗原,待测PRP作为竞争抗原,与Hib抗血清反应,建立间接竞争ELISA检测方法。采用棋盘法确定最佳包被多糖抗原浓度及Hib抗血清稀释倍数,绘制标准曲线,并确定最低检测限;对建立的方法进行批内和批间精密性验证;取Hib发酵液,在培养温度、接种量等因素相同的情况下,分别将培养液初始p H调至6.0±0.02、7.0±0.02和8.0±0.02,培养不同时间取样,采用建立的方法检测培养液内PRP含量。结果 PRP-Ty的最佳包被浓度为1.25μg/ml,Hib抗血清的最佳稀释倍数为300倍。该方法检测的线性范围为6.25~200 ng/ml,最低检测限为6.25 ng/ml,回归方程为y=-23.12 x＋99.62,R2=0.996。采用该方法分别重复检测3次高浓度（200 ng/ml）和低浓度（20 ng/ml）PRP多糖的变异系数（CV）分别为1.608%和3.344%;采用该方法及传统化学检测法分别检测6次同一批Hib成品分装疫苗的多糖,该方法检测结果的CV值为5.75%,传统化学法检测结果的CV值为1.98%,均符合要求;该方法检测不同初始p H培养液培养的Hib发酵液,培养时间为8 h左右时,PRP产量最高;当初始p H为8.0±0.02时,培养液内PRP含量增长最快,所达到的浓度最高。结论建立了Hib多糖间接竞争ELISA检测方法,该方法灵敏度较高,精密性良好,可应用于Hib多糖含量的定量检测。

Objective To develop, verify and preliminarily apply an indirect competitive ELISA method for determination of Haemophilus influenzae type b（Hib）polysaccharide. Methods An indirect competitive ELISA method was developed using Hib polysaccharide-tyramine（PRP-Ty）as coating antigen and PRP as competitive antigen for reaction with antisera against Hib. The polysaccharide antigen concentration and dilution of antisera against Hib were optimized by chessboard assay, based on which a standard curve was plotted, and the minimum detection limit was determined. The developed method was verified for precision in intra- and inter-assays. Hib fermentation liquid, of which the initial p H value was adjusted to（6. 0 ± 0. 02）,（7. 0 ± 0. 02） and（8. 0 ± 0. 02） respectively at the same culture temperature and inoculums,was cultured for various time periods. Samples were taken at various time points and determined for PRP content. Results The optimal coating concentration of PRP-Ty and dilution factor of antisera against Hib were 1. 25 μg / ml and 300 respectively. The linear range and minimum detection limit of the developed method were 6. 25 ~ 200 ng / ml and6. 25 ng / ml respectively, while the regression equation was y =-23. 12 x ＋ 99. 62, with a R2 value of 0. 996. The PRP at high（200 ng / ml）and low（20 ng / ml）concentrations were determined by the developed method, and the coefficients of variation（CVs）of results were 1. 608% and 3. 344%, respectively. The CVs of six determination results of polysaccharide contents in the same batch of final product of Hib by the developed method and traditional chemical test were 5. 75% and1. 98% respectively, which met the relevant requirements. The fermentation liquids of Hib at various initial p H values were determined by the developed method, and the result showed that the yield of PRP reached a peak value when the culture time was about 8 h. When the initial p H value was 8. 0 ± 0. 02, the PRP content in medium increased rapidly and reached the maximum concentration. Conclusion An indirect competitive ELISA method for determination of Hib polysaccharide was developed, which showed high sensitivity and precision, and might be used for the quantitative deter-mination of Hib polysaccharide content.