百日咳毒素抗原检测系统的建立及其应用

Development and application of pertussis toxin antigen detection system

目的建立吸附无细胞百白破联合疫苗中百日咳毒素(pertussis toxin,PT)的ELISA测定方法,并进行验证及应用。方法以PT作为检测抗原,用高效价鼠单抗和相应的兔多抗建立双抗体夹心ELISA,确定方法的线性范围、重复性、准确性、专属性等,并进行初步应用。结果 PT质量浓度在2.5～80.0 ng/m L时线性良好,决定系数R2>0.98。该方法与Ⅰ、Ⅱ、Ⅲ型脊髓灰质炎病毒原液、甲肝疫苗原液、乙肝疫苗原液、白喉类毒素(diphtheria toxoid,DT)、破伤风类毒素(tetanus toxoid,TT)、百日咳丝状血凝素(filamantous hemagglutinin,FHA)、黏着素(pertactin,PRN)均无明显交叉反应,重复性好,专属性较强,其他均符合常规质控要求。该抗原检测系统对生产具有指导意义,并可以正确反映原液及成品中PT的稳定性。结论建立了PT双抗体夹心ELISA检测方法,为吸附无细胞百白破联合疫苗生产过程中PT含量的质量控制提供有效的技术手段。

Objective To develop an ELISA( enzyme-linked immunosorbent assay) in detection content of pertussis toxin( PT) in Diphtheria,tetanus and acellular pertussis combined vaccine,adsorbed( DTa P),the validation and priminary application for this mothed was carried out. Methods The double antibody sandwich ELISA was set up by using PT as antigen,a mouse monoclonal antibody with high titer as capture antibody and corresponding rabbit antibody as the signal antibody in verification of linear range,reproducibility,accuracy and specificity,ect for the method. Results When PT was detected in an range 2.5 ng/mL to 80 ng/mL,the detection showed a good linearity with a correlation R2>0.98. The detections had no obvious cross reaction with the contents of type Ⅰ,Ⅱ and Ⅲ of poliomyelitis,hepatitis A,hepatitis B,diphtheria toxoid( DT),tetanus toxoid( TT),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. This method could correctly indicate the stability for PT in bulk and final products. Conclusion The developed ELISA provided an efficient technical means in quality control of PT antigen content in DTa P formulated product.