抗百日咳毒素单克隆抗体的纯化及应用研究

Purification and application of monoclonal antibodies against pertussis toxin

目的纯化抗百日咳毒素(PT)的单克隆抗体(M cAb),并建立特异、准确的PT定量检测方法。方法采用辛酸-硫酸铵沉淀法和A蛋白亲和层析法纯化杂交瘤细胞腹水,并经阻断抑制试验筛选识别不同表位的M cAb,用于建立定量检测PT的ELISA方法。结果经SDS-PAGE分析,纯化M cAb的纯度均在90%以上,选择二株识别不同抗原位点M cAbs,应用于检测PT的双抗夹心ELISA方法,灵敏度为2.14μg/L,批内变异系数5.85%,批间变异系数9.27%,平均回收率为108.12%,应用该法测定了国内几大生产厂家送检的无细胞百日咳疫苗原液中PT含量。结论获得了纯度高的抗PT M cAb,建立了一种特异、准确的定量检测PT的ELISA方法,并用于无细胞百日咳疫苗原液中PT含量的检测,为无细胞百日咳疫苗的质量控制提供了有力的手段。

Objective To purify monoclonal antibody (McAb) against pertussis toxin (PT), and develop a method for quantitative analysis of PT. Method The asites were purified by precipitation of caprylic acid and ammonium sulfate and affinity chromatography. The blocking ELISA was used to screen McAb which recognized different antigenic site, and to established an ELISA for quantitative measurement of PT. Result The result of SDS-PAGE showed that the purity of McAb were above 90%. We selected two strains of McAbs which recognized different antigenic site to establish a double antibody sandwich ELISA for measurement of PT, with a sensitivity of 2.14μg/L.The intra- and inter-assay coefficient of variation(CV) were 5.85%and 9.27% .The average recovery rates of the recovery assay was 108.12%. The method was applied for quantitative analysis pertussis toxin in bulk of acellular pertussis vaccine which were from severel factories. Conclusion Four high purified McAbs were obtained. An ELISA method for detecting quantitatively PT was established, which was applied for quantitative analysis pertussis PT in bulk of acellular pertussis vaccine and provided an useful tool for qulity control of acellular pertussis vaccine.