百日咳毒素兔多克隆抗体的制备及其ELISA定量检测方法的建立和验证

Preparation of rabbit polyclonal antibody and development and identification of quantitative ELISA detection method for pertussis toxin

目的制备百日咳毒素(pertussis toxin,PT)兔多克隆抗体(简称多抗),建立定量检测PT抗原含量的双抗体夹心ELISA法,并进行验证及应用。方法采用传统方法,用PT免疫青紫蓝兔制备PT兔多抗。优化ELISA系统反应条件,建立PT双抗体夹心ELISA定量检测方法,并进行特异性、线性、准确性、精密性、灵敏度验证。采用建立的方法对脱毒过程样品及其他百日咳抗原纯化过程样品菌毛蛋白(fimbriae proteins,FIM)原液中PT抗原含量进行检测。结果PT抗原含量双抗体夹心ELISA检测方法的工作条件为PT兔多抗包被浓度为1μg/mL,酶标抗体稀释度为1∶8000。此检测系统能与PT纯化蛋白发生特异性反应,而与百日咳丝状血凝素、白喉类毒素、破伤风类毒素均未发生交叉反应;建立的双抗体夹心ELISA法线性检测范围在25~400 ng/mL之间;PT高、中、低浓度回收率分别为103.27%、91.48%、103.52%;酶标板内和板间的变异系数(CV)均小于10%;该方法的灵敏度为20.719 ng/mL,检测下限为41.438 ng/mL。检测5种不同脱毒工艺条件下、不同取样时间点的35批样品,其抗原含量变化均符合脱毒反应变化趋势。结论成功制备了PT兔多抗,并建立了精密性和准确性良好的定量检测PT抗原含量的双抗体夹心ELISA法,可用于组分百白破联合疫苗生产过程中化学脱毒样品的抗原含量检测。

Objective To prepare rabbit polyclonal antibodies against pertussis toxin(PT)and develop a double antibody sandwich ELISA for quantitative determination of PT antigen,identify and apply the method.Methods The rabbit polyclonal antibody against PT was prepared by immunizing Chinchilla rabbit with PT using traditional method.The reaction conditions of ELISA system were optimized,the double antibody sandwich ELISA method for quantitative determi-nation of PT was developed,and the specificity,linearity,accuracy,precision and sensitivity were verified.The developed method was used to detect PT antigen content in fimbriae proteins(FIM)stock solution of samples during detoxification and other purification process of pertussis antigen.Results The working condition of double antibody sandwich ELISA for detection of PT antigen content was the coating concentration of PT rabbit polyclonal antibody of 1μg/mL,and the enzyme-labeled antibody dilution of 1:8000.This detection system showed specific reaction with PT purified protein,but had no cross reaction with filamentous hemagglutinin,diphtheria toxoid and tetanus toxoid;the linear detection range of the developed double antibody sandwich ELISA was within 25—400 ng/mL;the recovery rates of PT at high,moderate and low concentrations were 103.27%,91.48%and 103.52%,respectively;both the intra-and inter-coefficients of variation(CVs)were less than 10%;the sensitivity of the method was 20.719 ng/mL,and the detection limit was 41.438 ng/mL.Thirty-five batches of samples were detected under five different detoxification process conditions and at different sampling time points,and the changes of antigen content were all consistent with the trend of detoxification reaction.Conclusion The PT rabbit polyclonal antibody was successfully prepared,and a double antibody sandwich ELISA with high precision and accuracy was developed for the quantitative determination of PT antigen content,which can be used for the antigen content detection of chemically detoxified samples in the production process of component DPT vaccines.