检测百日咳抗原的系列酶免疫测定试剂盒的稳定性及其在百日咳疫苗生产中的应用

Stability of EIA kits for determination of pertussis antigens and their application in the production process of pertussis vaccine

目的 评估检测百日咳毒素（pertussis toxin,PT）、丝状血凝素（filamentous haemagglutinin,FHA）和黏着素（pertactin,Prn）的3种酶免疫测定试剂盒的稳定性及其在无细胞百日咳疫苗（acellular pertussis vaccine,aPV）生产质量控制中的应用.方法 对3种酶免疫测定试剂盒进行热加速试验（37℃放置3 d）和长期稳定性试验（4℃放置6月）,根据试剂盒的标准曲线相关系数、最高浓度标准品与阴性对照的吸光度值之比（P/N）值、质控品回收率评估试剂盒的稳定性.将3种试剂盒用于aPV生产的发酵、纯化、吸附阶段的PT、FHA、Prn定量检测,以确定3种试剂盒对aPV生产质量控制的可行性.结果 3种酶免疫测定试剂盒的37℃热加速试验和4℃长期稳定性试验的各项质量参数均符合相关要求.采用试剂盒定量各生产阶段的百日咳抗原含量显示：百日咳杆菌的发酵终点在第42～46 h;各3批PT、FHA和Prn组分液的纯化回收率分别为49.24％、56.12％和63.65％,68.75％、55.60％和49.76％、75.73％、60.63％和50.10％.采用单独吸附的铝佐剂抗原吸附率高于采用混合吸附,但单独和混合吸附方式制备的疫苗的效力无差异.结论 检测PT、FHA和Prn的3种酶免疫测定试剂盒具有良好的稳定性,可用于aPV生产的质量控制.

Objective To evaluate the stability of 3 kinds of EIA kits for detecting pertussis toxin （PT）,filamentous haemagglutinin （FHA） and pertactin （Prn） and their application in the quality control of acellular pertussis vaccine （aPV） production.Methods The stability of EIA kits were evaluated by heat accelerated test （37 ℃ for 3 days） and long-term stability test （4 ℃ for 6 months）,using the correlation coefficient of standard curve,the P/N value and the recovery of the quality control material.The contents of PT,FHA and Prn in the fermentation,purification and adsorption stages during the production process of aPV were detected by these 3 kinds of EIA kits,and the feasibility of using them in the quality control of aPV production were determined.Results Heat accelerated stability test and long-term stability test showed that quality parameters of 3 kinds of EIA kits all met the requirement.The detection results of pertussis antigens in the different production stages of pertussis vaccine obtained by 3 kinds of EIA kits showed that the fermentation end of Bordetella pertussis was at 42-46 h,and the purification recovery rates were 49.24%,56.12% and 63.65% for 3 lots of PT antigen,respectively,68.75%,55.60% and 49.76% for 3 lots of FHA antigen,respectively,and 75.73%,60.63% and 50.10% for 3 lots of Prn antigen,respectively.The antigen absorption rates of aluminum adjuvant with separate absorption were higher than those with mixed adsorption,but there was no difference in potency between both vaccines prepared by separate and mixed adsorption.Conclusion Three kinds of EIA kits for detecting PT,FHA and Prn have good stability and can be used for the quality control of aPV production.