The Establishment And Verification Of Detective Protocol For Residual Protein Of Influenza Virus Host Cell MDCK

[Background]The wide spread of influenza virus has brought heavy economic and social burden to all parts of the world.Influenza vaccination is an effective way to prevent infection of influenza virus.Canine kidney cells MDCK(madin darby canine kidney)are adherent cells isolated from the kidney tissue of normal adult cocker spanies.These cells are sensitive to influenza virus and have high application value in the research and production of cell matrix influenza virus vaccine.When using MDCK cell matrix to produce influenza vaccine,HCP(host cell protein)is usually retained in the vaccine product.The adverse reactions caused by the residual HCP in the vaccine may seriously affect the safety and efficacy of the vaccine product.In order to ensure the product quality,it is necessary to effectively control the HCP level.Therefore,a sensitive and stable detection method is needed to detect the residual HCP in the vaccine.[Objective]To establish and verify a double antibody sandwich ELISA method for detection of HCP in MDCK cell matrix 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 purified,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,so as to establish a double antibody sandwich ELISA method for the detection of MDCK HCP.Determine the working concentration antibodies used in this method and its linear range,and verify the accuracy,repeatability,intermediate precision,durability and specificity.Then,the samples in the production process are verified by this method,and the test results are compared with those of the commercial kit.[Results]The reference substance prepared by two-step chromatography is mixture of many kinds of proteins,the size is distributed between 25-80k Da,and the total protein concentration is 31.74?g/m L.High quality antibodies were obtained by immunizing New Zealand rabbits and Boer goat,the titers of the antibodies were 1:512000 and 1:128000respectively.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 ELISA method.The square matrix titration determined that the working concentration of the coated antibody was 4?g/m L,and the working concentration of the antibody was 1:3200.The linear relationship of the method was R~2>0.99 in the range of 20?400 ng/m L,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%.This method is used to detect the production samples,and statistical results have no differences.[Conclusion]A double antibody sandwich ELISA method for the detection of residual HCP in MDCK cell matrix influenza vaccine was established.The methodological verification result of this method is good.It can be used for the detection of residual HCP in MDCK cell matrix influenza vaccine production,and is of great significance for the quality control of vaccine production.