MDCK细胞宿主蛋白含量双抗体夹心ELISA检测方法的建立

Developing a double-antibody sandwich ELISA in determination of host cell protein from MDCK cell

目的建立检测犬肾细胞(madin-darby canine kidney, MDCK)宿主细胞蛋白(host cell protein, HCP)含量的双抗体夹心ELISA。方法从MDCK细胞中提取细胞总蛋白,免疫新西兰兔制备兔抗MDCK细胞蛋白多克隆抗体,抗体经辛酸-硫酸铵沉淀和Protein A层析纯化后,采用SDS-PAGE分析抗体纯度,Western blot检测抗体特异性。用纯化的多克隆抗体作为包被抗体,并采用改良过碘酸钠标记法制备酶标抗体,建立ELISA并确定包被抗体浓度和辣根过氧化物酶(HRP)标记抗体稀释度等最适条件。确定该方法较佳的线性范围及检测限,并对该法特异性、准确度、精密性和重复性进行验证。最后,用该方法分别对接种流感病毒的MDCK细胞上清收获液和纯化样品进行MDCK细胞蛋白含量检测,初步验证其在纯化工艺开发中的适用性。结果通过免疫新西兰兔制备了高滴度的兔抗MDCK细胞蛋白多克隆抗体血清,滴度可达1∶8 000。纯化后的兔抗MDCK细胞蛋白多克隆抗体纯度>90%,并可与MDCK细胞蛋白特异性结合。建立的双抗体夹心ELISA的理想包被抗体质量浓度为10μg/mL,酶标抗体的工作浓度为1∶500稀释。该方法的线性范围为50~2 500 ng/mL,检测限为50 ng/mL;该方法对Vero细胞、293T细胞和Mrc-5细胞等其他细胞HCP无交叉反应,特异性良好;不同浓度的MDCK细胞HCP回收率在98.5%~111.9%之间,变异系数均<10%。接种流感病毒的MDCK细胞培养上清经多步纯化后MDCK细胞蛋白质量浓度逐渐降低至<900 ng/mL,纯化工艺可有效去除MDCK细胞蛋白残留。结论建立双抗体夹心ELISA检测MDCK细胞残余HCP含量的方法,可用于基于MDCK细胞培养的流感疫苗下游工艺开发中宿主细胞残留HCP含量监测。

Objective To develop a double-antibody sandwich ELISA in determination of host cell protein(HCP) from MDCK cell. Methods Total cell protein was extracted from MDCK cell and used to immunize rabbits to prepare polyclonal antibodies. The rabbit serum antibodies were purified by octanoic acid-ammonium sulfate precipitation and then by protein A affinity chromatography. The SDS-PAGE and Western-blot were used to identify the purity and specificity of the purified antibodies. In development of a double antibody sandwich ELISA, the purified polyclonal antibodies were both used as capture antibody and signal antibody labeled with HRP by modified sodium periodate labeling method. The optimal conditions, including the concentration of capture antibody and the dilution of HRP-labeled antibody were optimized. The optimal linear range and minimum detection limit of the method were determined, and the specificity, accuracy and repeatability were verified. Finally, a preliminarily verification of its applicability in preparation of MDCK cell derived influenza vaccine was performed, and the HCP in the harvested supernatant of influenza virus infected MDCK cells and purified samples were determined by the developed method. Results The rabbit polyclonal antibodies with high titers against MDCK cell protein were prepared by immunized rabbits, with a titer of up to 1∶8 000. The purified antibodies reached a purity of more than 90%, and could specifically bind to MDCK cell proteins. The optimal working concentrations for capture antibodies and signal antibodies were 10 μg/mL and 1∶500 dilution, respectively. The optimal linear range of the method was 50-2 500 ng/mL while the minimum detection limit was 50 ng/mL. The method had a good specificity and no cross-reaction with HCPs from Vero cell, 293 T cell and Mrc-5 cell. The recovery rates for MDCK cell protein antigens at different concentrations were between 98.5%-111.9%, with a coefficient of variation less than 10%(1.5%-7.2%). After multi-step purification, the HCP contents in the harvested supernatant from MDCK cell inoculated with influenza virus was gradually reduced to less than 900 ng/mL, thus HCP can be effectively removed in the downstream process. Conclusion A double antibody sandwich ELISA in detection of residual HCP from MDCK cells was successfully developed, which could be used in determination of residual HCP content in downstream process in preparation of MDCK cell derived influenza vaccine.