百日咳菌毛抗原检测方法的建立及初步应用

Establishment and preliminary application of pertussis fimbriae detection scheme

目的建立百日咳菌毛(fimbriae, Fim)抗原组分纯化过程中含量监测的双抗体夹心ELISA方法。方法 Fim抗原免疫家兔获得多克隆抗血清,经辛酸-硫酸铵法纯化并用辣根过氧化物酶标记,采用棋盘滴定法确定最佳包被抗体浓度和酶标抗体最适稀释倍数,建立双抗体夹心ELISA检测法,并对其进行全面验证。结果棋盘滴定法确定包被抗体浓度为2μg/mL,最适酶标记抗体稀释倍数为2 000倍。验证双抗体夹心ELISA线性检测范围为15.625~250.000 ng/mL(相关系数r>0.99)。该方法与Ⅰ、Ⅱ、Ⅲ型脊髓灰质炎病毒抗原、白喉类毒素(diphtheria toxin, DT)、破伤风类毒素(tetanus toxin, TT)、b型流感嗜血杆菌荚膜多糖(Hib)、百日咳毒素(pertussis toxin, PT)、百日咳丝状血凝素(filamentous haemagglutinin, FHA)和百日咳黏着素(pertactin, PRN)抗原均无明显交叉反应,重复性好,专属性强,其他均符合常规质控要求。结论建立了Fim双抗体夹心ELISA检测法,为百日咳菌毛蛋白生产过程和以组分百日咳疫苗为基础的联合疫苗中Fim含量的质量控制提供有效技术手段。

Objective To develop an ELISA method for quantitative determination of pertussis fimbriae durning purification of pertussis antigen components. Methods Rabbits were immunized with Fim antigen to obtain polyclonal antiserum against Fim. The polyclonal antiserums were purified by octanoic acid-ammonium sulfate method and then were labeled with horseradish peroxidase. The best concentration of coated antibody and the optimal dilution ratio of enzyme labeled antibody were determined by chessboard titration. The established double-antibody sandwich ELISA method was fully verified. Results The optimal concentration of coated antibody and dilution ratio of enzyme labeled antibody were 2 μg/mL and 2000 times, respectively. The best linearity of the ELISA method was found in a range of 15.625-250.000 ng/mL(r>0.99). The detections had no obvious cross reaction with the contents of type I, II and III of poliomyelitis, diphtheria toxoid(DT), tetanus toxoid(TT), Haemophilus in?uenzae type b(Hib), pertussis toxin(PT), filamentous hemagglutinin(FHA) and pertactin(PRN), and with good repeatability and strong specificity, other aspects were all in accordance with the requirements of the routine quality control. Conclusion The established ELISA method can be applied to detect Fim during purification of pertussis antigen components, which will be as an efficient technical means in quality control of Fim antigen content in DTaP formulated product.