酿酒酵母残留蛋白ELISA定量检测方法的建立

目的建立酿酒酵母残留蛋白(Host cell protein,HCP)ELISA定量检测方法,为重组乙型肝炎疫苗(酿酒酵母)及相关生物制品的质量控制提供依据。方法制备酿酒酵母全细胞蛋白参考品,以其为抗原,免疫家兔,采用辛酸-硫酸铵沉淀法纯化抗体,SDS-PAGE分析纯度,免疫双向扩散法测定抗体效价,Western blot法分析抗体的特异性。以制备的抗酿酒酵母多克隆抗体作为包被抗体,HRP标记的抗酿酒酵母多克隆抗体作为检测抗体,建立酿酒酵母抗原双抗体夹心ELISA定量检测方法;梯度稀释酿酒酵母蛋白抗原参考品,并绘制标准曲线,确定该方法的线性范围及检测限,并进行特异性、准确度、精密度及适用性验证。结果共制备3批酿酒酵母抗原蛋白,其平均含量分别为1.61、1.64和1.63 mg/ml;纯化的IgG抗体纯度为91.7%,抗体效价为1∶64,能与酿酒酵母蛋白多数条带结合;酿酒酵母蛋白抗原浓度在6.25～200 ng/ml的范围内,线性关系良好(R2>0.99),最低检测限为6.25 ng/ml;用建立的方法检测小牛血清、人血白蛋白、MEM培养基、Vero细胞上清蛋白、Vero细胞乙型脑炎疫苗及PHK细胞狂犬疫苗,均无交叉反应,特异性良好;检测不同浓度的抗原回收率为97.6%～107.00%,变异系数均<10%,最低定量检测限为25 ng/ml;检测3批酿酒酵母重组乙型肝炎疫苗生产各工序中酿酒酵母蛋白的残留量,能有效反映杂蛋白去除率。结论成功建立了酿酒酵母HCP双抗体夹心ELISA定量检测方法,该方法特异性强,精密度及准确度良好,可用于生物制品中酿酒酵母HCP的检测。

Development of an ELISA kit for host cell protein of Saccharomyces cerevisiae

Objective To develop an ELISA kit for host cell protein(HCP)of Saccharomyces cerevisiaes and provide a basis for quality control of recombinant hepatitis B vaccine(S.cerevisiae)and ther relevant biologics.Methods Whole cell protein reference of S.cerevisiae was prepared and as an antigen to immunize rabbits.The antibody was purified by caprylic acid-ammonium sulfate precipitation method,then determined for purity by SDS-PAGE,for antibody titer by double immunodiffusion test,and for specificity by Western blot.A double antibody sandwich ELISA method for quantitative determination of S.cerevisiae was developed using the prepared polyclonal antibody against S.cerevisiae as coating antibody and HRP-labeled polyclonal antibody against S.cerevisiae as enzyme labeled antibody.The S.cerevisiae protein antigen was diluted serially to plot a standard curve,based on which the developed method was determined for linear range and detection limit,and verified for specificity,accuracy,precision and suitability.Results Three batches of S.cerevisiae antigen protein were prepared,of which the mean protein contents were 1.61,1.64 and 1.63 mg / ml respectively.The purified IgG reached a purity of 91.7% and a titer of 1 ∶ 64,which showed specific bindings to several S.cerevisiae protein bands.The developed ELISA method showed good linearity within a range of S.cerevisiae protein antigen concentration of 6.25 ～ 200 ng / ml(R2 > 0.99),of which the minimum detection limit was 6.25 ng / ml.No cross reactions with calf serum,human serum albumin,MEM,supernatant of Vero cells,purified Japanese encephalitis vaccine or freeze-dried rabies vaccine for human use were observed,indicating high specificity of the developed ELISA method.The recovery rates of antigen at various concentrations determined by the method were 97.6% ～ 107.00%,with coefficients of variation of less than 10%.The minimum quantitative detection limit of the method was 25 ng / ml.The developed ELISA method was used for determination of residual S.cerevisiae protein contents in three batches of recombinant hepatitis B vaccine during production,which reflected the removal rate of foreign protein effectively.Conclusion A double antibody sandwich ELISA kit for determination of HCP of S.cerevisiae content was developed,which showed high specificity,precision and accuracy,and might be used for determination of HCP of S.cerevisiae in biologics.