百日咳丝状血凝素单克隆抗体的制备及ELISA定量检测方法的建立

Preparation of Monoclonal Antibody and Development of Quantitative ELISA for Pertussis Filamentous Hemagglutinin

目的制备百日咳丝状血凝素(Filamentous hemagglutinin,FHA)单克隆抗体,并建立双抗体夹心ELISA法用于检测FHA的含量。方法采用杂交瘤技术制备FHA单抗,并对其进行鉴定;优化ELISA系统反应条件,建立FHA双抗体夹心ELISA定量检测方法,并对其进行精密性及准确性验证;应用建立的方法检测本所12批无细胞百日咳疫苗生产中间样品中FHA的含量。结果获得4株稳定分泌抗FHA单抗的杂交瘤细胞株,抗体亚型分别为IgG1和IgG2b,抗体腹水ELISA效价达1∶105～1∶106,均能与FHA发生特异性反应,而与百日咳毒素和流感病毒血凝素未发生交叉反应;建立的双抗体夹心ELISA法的线性检测范围在1.56～100.00 ng/ml之间,板内和板间变异系数均小于10%;FHA高、中、低浓度的回收率分别为94.73%、108.67%和116.37%;12批无细胞百日咳疫苗生产中间样品中FHA的含量在X±2SD范围内比较稳定。结论已成功制备了抗FHA单抗,并建立了精密性和准确性均较好的定量检测FHA的双抗体夹心ELISA法,可用于无细胞百日咳疫苗生产中间产品中FHA的定量检测。

Objective To prepare monoclonal antibody（McAb） and develop an ELISA method for quantitative determination of pertussis filamentous hemagglutinin（FHA）.Methods The McAbs against FHA was prepared by hybridoma technique and identified.The reaction condition of ELISA system was optimized,based on which double antibody sandwich ELISA for quantitative determination of FHA was developed and verified for precision and accuracy.The FHA contents in 12 batches of intermediate products of acellular pertussis vaccine were determined by the developed ELISA method.Results Four hybridoma cell strains stably secreting McAb against FHA were obtained,and the secreted McAbs were of IgG1 and IgG2b subtypes,with an ELISA titer of 1 ∶ 105 ～ 1 ∶ 106 in ascites,and showed specific reaction with FHA,while showed no cross reaction with pertussis toxin or influenza virus hemagglutinin.The linear detection range of developed ELISA method was 1.56 ～ 100.00 ng / ml,while both the intra-and inter-coefficients of variation were less than 10%.The recovery rates of FHA at high,moderate and low concentrations were 94.73%,108.67% and 116.37% respectively.The FHA contents（X ± 2SD） in 12 batches of intermediate products of acellular pertussis vaccine were stable.Conclusion The McAbs against FHA of pertussis were successfully prepared,and a double antibody sandwich ELISA method with high precision and accuracy was developed,which might be used for the determination of FHA content in intermediate products of acellular pertussis vaccine.