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目的建立基于Luminex技术的多组分百日咳疫苗血清抗体效价快速检测方法。方法将百日咳类毒素(pertussis toxin,PT)、丝状血凝素(filamentous haemagglutinin,FHA)、百日咳杆菌黏附素(pertactin,PRN)、百日咳菌鞭毛蛋白2,3型(fimbrial-2 and fimbrial-3,FIM 2,3)分别与4种不同型号的微球耦联,耦联的抗原在同一孔中与血清孵育,通过生物素标记抗体和链霉素标记藻红蛋白双重识别,用Luminex荧光检测得到荧光中位数值(MFI),进行效价分析,并通过百日咳标准血清建立五组分百日咳疫苗血清抗体检测的标准曲线。采用ELISA法检测同一组血清样品中的抗体效价,并对两种方法的检测结果进行比较。结果与ELISA法相比,Luminex法的线性范围更宽。两种方法检测五组分百日咳疫苗血清抗体效价的分布范围接近,Luminex法检测50份五组分百日咳疫苗免疫小鼠血清抗体效价的平均值分别为PT 65.365 IU/ml、FIM 15.052 IU/ml、FHA 545.236 IU/ml、PRN 7.876 IU/ml,ELISA法检测的平均值分别为PT 72.640 IU/ml、FIM 16.480 IU/ml、FHA 589.474 IU/ml、PRN 7.345 IU/ml,两种检测方法的检测结果具有良好的相关性,相关系数(R2)分别为:PT 0.905 8、FIM 0.979 1、FHA 0.930 9、PRN 0.997 6。结论与ELISA法相比,基于Luminex的多重分析物的检测方法具有减少样品消耗量、节省成本和测定时间、使多种目标分子之间相关性的分析更准确等优点,可用于五组分百日咳疫苗血清抗体效价的检测。
OBJECTIVE To establish a rapid method for the detection of serum antibody titer of the multicomponent pertussis vaccine based on Luminex technology. Methods Pertussis toxin (PT), filamentous hemagglutinin (FHA), pertactin (PRN), fimbrial-2 and fimbrial-3 , FIM 2,3) were coupled to four different types of microspheres, respectively. The coupled antigen was incubated with serum in the same well and double-labeled with biotin-labeled antibody and streptomycin-labeled phycoerythrin. Luminex fluorescence Fluorescence median (MFI) values were obtained for potency analysis and a standard curve for the five-component pertussis vaccine serum antibody test was established by pertussis standard serum. Antibody titers in the same group of serum samples were measured by ELISA and the results of the two methods were compared. Results The Luminex method had a wider linear range than the ELISA method. The range of distribution of the antibody titers of five components of pertussis vaccine was close to that of the two methods. The average antibody titers of 50 immunized mice with pertussis vaccine by Luminex were PT 65.365 IU / ml, FIM 15.052 IU / ml, FHA 545.236 IU / ml and PRN 7.876 IU / ml, respectively. The mean value of ELISA was 72.640 IU / ml, FIM 16.480 IU / ml, FHA 589.474 IU / ml and PRN 7.345 IU / The correlation coefficients (R2) were: PT 0.905 8, FIM 0.979 1, FHA 0.930 9, PRN 0.997 6, respectively. Conclusion Compared with ELISA, the detection method of multiple analytes based on Luminex has the advantages of reducing sample consumption, saving cost and measuring time, making the analysis of the correlation between multiple target molecules more accurate and so on, and can be used for five-component pertussis vaccine Detection of serum antibody titer.