Sf9昆虫细胞宿主蛋白含量双抗体夹心ELISA检测方法的建立

Development of double antibody sandwich ELISA for host cell protein content of Sf9 insect cells

目的建立Sf9昆虫细胞宿主蛋白（host cell protein,HCP）含量双抗体夹心ELISA检测方法。方法从Sf9昆虫细胞中提取总蛋白,免疫家兔,制备兔抗Sf9细胞总蛋白多克隆抗体,经辛酸-硫酸铵沉淀和CL-4B层析柱纯化后,采用SDS-PAGE分析抗体纯度,间接ELISA法检测抗体效价,Western blot法检测抗体特异性;用纯化的多克隆抗体作为包被抗体,采用改良过碘酸钠法将HRP酶标记至纯化的抗体上作为酶标抗体,采用方阵滴定法确定双抗体夹心ELISA方法的最适工作条件。确定该方法的最佳线性范围及最低检测限,验证该方法的准确度及精密度。将表达戊型肝炎ORF2基因的重组杆状病毒感染Sf9细胞,收获病毒培养上清,制备3批纯化的重组蛋白,用建立的方法检测纯化过程中HCP含量的变化,验证其在纯化工艺中的适用性。与商品化试剂盒进行比较,检测两种方法的灵敏度及在制品中的适用性。结果纯化后的兔抗Sf9细胞总蛋白多克隆抗体纯度达90%以上,抗体效价为1∶10 000,可与Sf9细胞蛋白特异性结合。建立的双抗体夹心法ELSA方法最佳抗体包被浓度为20μg/ml,37℃孵育1 h;酶标抗体的工作浓度为1∶200,37℃孵育1 h;TMB室温显色30 min;测定各孔A450值。该方法的最佳线性范围为50～1 600 ng/ml,最低检测为50 ng/ml;不同浓度的Sf9细胞蛋白抗原回收率在87.8%～117%之间,变异系数均小于10%;制备的3批重组蛋白经超滤及层析纯化后,HCP含量均逐渐降低至小于50 ng/ml,纯化工艺可有效去除HCP;与商品化试剂盒比较,该方法更适用于检测戊肝类病毒颗粒样品。结论已成功建立Sf9昆虫细胞残余蛋白含量检测的双抗体夹心法ELSA方法,可用于检测Sf9昆虫细胞/杆状病毒表达系统纯化的重组蛋白中HCP含量。

Objective To develop a double antibody sandwich ELISA for host cell protein（HCP）content of Sf9 insect cells. Methods Total protein was extracted from Sf9 insect cells,with which rabbits were immunized. The prepared polyclonal antibody was purified by caprylic acid-sodium caprylate precipitation and CL-4B chromatography, then determined for purity by SDS-PAGE,for titer by indirect ELISA,and for specificity by Western blot. A double antibody sandwich ELISA was developed using the purified polyclonal antibody as a coating antibody,and the purified polyclonal antibody labeled with HRP by modified sodium periodate method as enzyme-labeled antibody,of which the working condition was optimized by block titration,while the optimal linear range and minimum detection limit were determined, and the accuracy and precision were verified. Sf9 cells were infected with recombinant baculovirus expressing hepatitis E virus ORF2 gene,of which the culture supernatant was collected and prepared into three batches of purified recombinant protein. The change of HCP content during purification was determined by the developed method to verify the suitability. The sensitivity and suitability of the developed method were compared with those of commercial kit. Results The purity of polyclonal antibody against total protein of Sf9 cells reached a purity of more than 90% and a titer of 1 ∶ 10 000,which showed specific binding to Sf9 cell protein. The optimal antibody concentration for coating in the developed double antibody sandwich ELISA was 20 μg / ml,and the samples were incubated at 37 ℃ for 1 h. The working concentration of enzyme-labeled antibody was 1 ∶ 200,while the samples were also incubated at 37 ℃ for 1 h. The time for color development with TMB at room temperature was 30 min. The optimal linear range of the developed method was 50 ～ 1 600 ng / ml,while the minimum detection limit of 50 ng / ml. The recovery rates of Sf9 cell protein antigen at various concentrations was 87. 8% ～ 117%,with a coefficient of variation of less than 10%. The HCP contents in three batches of prepared recombinant protein decreased gradually to less than 50 ng / ml,indicating that HCP was effectively removed. Compared with commercial kit, the developed method was more suitable for determination of hepatitis E virus-like particles. Conclusion A double antibody sandwich ELISA for residual host cell protein of Sf9 insect cells was successfully developed,which might be used for determination of HCP content in purified recombinant protein expressed in Sf9 insect cells / baculovirus expression system.