流感疫苗工艺特异性MDCK宿主蛋白ELISA检测方法的建立与应用

Establishment and application of ELISA detection protocol of process-specific MDCK host protein of influenza vaccine

目的建立一种能够检测MDCK细胞基质流感疫苗中宿主细胞蛋白(host cell protein,HCP)含量的双抗体夹心ELISA并进行验证。方法通过培养MDCK细胞获取工艺特异性的MDCK HCP,进一步处理获得参考品。将MDCK HCP分别免疫新西兰兔和波尔山羊获得抗HCP的多克隆抗体。抗体经纯化后进行SDS-PAGE分析,间接ELISA分析鉴定抗体效价并验证特异性,选择效价较高的兔抗体作为包被抗体,使用HRP标记羊抗体获得显示抗体,从而建立检测MDCK HCP的双抗体夹心ELISA。确定该方法所用抗体的工作浓度以及线性范围,验证准确性、重复性、中间精密度、耐用性和特异性。然后,使用该方法对生产过程中的样品进行检定,并将检测结果与商品试剂盒的检测结果进行比对。结果制备的HCP参考品相对分子质量分布在25000~80000之间,抗原质量浓度为31.74μg/mL。免疫新西兰兔和波尔山羊获得高质量抗体,抗体效价分别为1∶512000、1∶128000,纯化后的多抗特异性良好。选择抗体效价较高的兔抗体作为包被抗体,工作浓度较高的酶标羊抗作为显示抗体建立双抗体夹心ELISA。方阵滴定法确定了包被抗体工作质量浓度为4μg/mL,显示抗体的工作浓度为1∶3200,该方法在20~400 ng/mL范围内线性关系良好(R^(2)>0.99),不同浓度的MDCK HCP回收率在95.62%~113.82%之间,CV<10%,重复性和中间精密度结果的CV<10%,显示抗体孵育时间在0.5~1.5 h内对实验无影响,显色时间在10~20 min内对实验无影响,特异性良好。使用该方法对生产样品进行检测,与商用试剂盒的检测结果差异无统计学意义(P=0.236,P>0.05)。结论建立了能检测MDCK细胞基质流感疫苗中残留HCP的双抗体夹心ELISA,方法学验证结果良好,可用于MDCK细胞基质流感疫苗生产中残留HCP的检定,对疫苗生产质量控制具有重要意义。

Objective To establish and verify a double antibody sandwich ELISA method for detection of HCP content in MDCK cell-based influenza vaccine. Methods The process-specific MDCK HCP was obtained by culturing MDCK cells and the reference material was obtained by further treatment. Polyclonal antibodies against MDCK HCP were obtained by immunizing New Zealand rabbits and Boer goat with HCP respectively. The antibody was identified by SDS-PAGE analysis after being purified, and the titer of the antibody and the specificity was verified by indirect ELISA analysis. The rabbit antibody with high titer was selected as the coating antibody and the goat antibody was labeled with HRP to obtain the display antibody to therefore establish a double antibody sandwich ELISA method for the detection of MDCK HCP. The working concentration of the antibodies used in this method and the linear range was determined, and their accuracy, repeatability, intermediate precision, durability and specificity were verified. Then, the samples in the production process were verified by this method, and the test results were compared with those of the commercial kit. Results The relative molecular weight of the HCP reference substance was distributed between 25 000-80 000, and the antigen concentration is 31.74 μg/mL. High quality antibodies were obtained by immunizing New Zealand rabbits and Boer goat, the titers of the antibodies were 1∶512 000 and 1∶128 000 respectively. The purified polyclonal antibodies had good specificity. Rabbit antibody with high titer was selected as coating antibody, and the goat antibody with high working concentration was used as the display antibody to establish the double antibody sandwich ELISA method. The square matrix titration determined that the working concentration of the coated antibody was 4 μg/mL, and the working concentration of the antibody was 1∶3 200. The linear relationship of the method was good in the range of 20-400 ng/mL(R;> 0.99). The recovery rate of MDCK HCP of different concentrations was between 95.62% and 113.82%. The coefficient of variation was less than 10%,and the coefficient of variation of repeatability and intermediate precision was less than 10%. The antibody incubation time had no effect on the experiment within 0.5 h and 1.5 h, and the chromogenic time had no effect on the experiment in 10-20 min;therefore the specificity was good. When this method was used to detect the residual HCP in the production samples, there was no significant difference between the test results by this method and the commercial kit(P=0.236, P>0.05). Conclusion A double antibody sandwich ELISA method for the detection of residual HCP in MDCK cell-based influenza vaccine is established. The methodological verification result of this method is good. It can be used for the detection of residual HCP in MDCK cell-based influenza vaccine production, and is of great significance for the quality control of vaccine production.