Establishment Of Double-antibodies Sandwich ELISA Detection Method For MDCK Cell Protein

MDCK(Madin-Darby canine kidney cell line)was isolated from an adult Cocker Spaniel in kidney of adherent cells.MDCK cells are sensitive to adenovirus,canine parvovirus,reovirus,avian influenza virus and many other viruses.Moreover,the cells are easy to be cultured on a large scale,the proliferation is fast,and influenza virus infection with MDCK cells is highly efficient and produces high titer.So MDCK cells are one of the cell lines producing influenza A and influenza B vaccines.The presence of host cell proteins in vaccines may be toxic to the body and may cause allergic reactions.In order to ensure the safety and efficacy of vaccine products,Chinese Pharmacopoeia limits the residue of host cell proteins.This study was a preliminary exploration of the detection methods of MDCK cell protein in the vaccine.In this study,MDCK cell protein antigen with content of 5.03 mg/mL was prepared by using MDCK cells.The polyclonal antibody was obtained by immunizing rabbits,guinea pigs,chickens and sheep.Because of the MDCK antibody levels were too low in guinea pig and chicken blood,the Rabbit anti MDCK and Goat anti MDCK polyclonal antibody were used to establish the double antibody sandwich ELISA method.Then the antibody titer was determined by indirect ELISA method.The level of MDCK antibody in rabbit blood was 1:10000,and in sheep blood was 1:1000.The titer of Rabbit anti-MDCK polyclonal antibody was 1:32 and Goat anti MDCK polyclonal antibody was 1:8 by double agar immunodiffusion test.Analysis by western blot,the Rabbit anti MDCK and Goat anti MDCK polyclonal antibody had no cross reaction with fetal bovine serum,newborn bovine serum and horse serum.The results showed that the polyclonal antibody prepared by this method had good specificity.The purified goat anti-MDCK polyclonal antibody was used as the coated antibody,Rabbit anti MDCK polyclonal antibody labeled with modified sodium periodate as enzyme labeled antibody,then the double antibody sandwich ELISA method for detection of MDCK cell protein was established.After the optimization of the reaction conditions,the optimal conditions obtained.The best concentration of Goat anti MDCK antibody was 10μg/mL,the best blocking solution was 10%horse serum,and the optimal dilution of enzyme labeled Rabbit anti MDCK antibody was 1:2000.MDCK cell protein content in the range of 2.5-40μg/mL had a good linear relationship.the minimum detection content was 2.5μg/mL.The quality evaluation results showed that the established double antibody sandwich ELISA method was specific,stable and reproducible.The double antibody sandwich ELISA method was used to detect the MDCK cells recombinant avian influenza vaccine(Re-6 strain +Re-7 strain + Re-8 strain)the residual protein could be detected.At present,the "Chinese Pharmacopoeia" has not been the relevant provisions on MDCK cell protein residues in the vaccine.In this study,established double antibody sandwich ELISA quantitative detection method laid the foundation of monitoring and testing for the product based on MDCK cells of human and veterinary vaccine.It also provided a guarantee of improving the quality of vaccine and reducing the risk of vaccine market.