Establishment and application of double antibody sandwich ELISA quantitative method for BCSP31 protein
In order to accurately quantify the effective content of the core component BCSP31 pro-tein of Brucella chimeric virus-like particle vaccine,a double-antibody sandwich ELISA method was established in this study.BCSP31 was prepared and purified by prokaryotic expression system as immunogen and standard substance,rabbits and BALB/c mice were immunized to prepare rab-bit-derived capture polyclonal antibody and mouse-derived detection polyclonal antibody.The pa-rameters of the double antibody sandwich ELISA method,such as the optimal dilution times of capture antibody and detection antibody,the type of blocking solution and blocking conditions and the working conditions of enzyme-labeled secondary antibody were optimized.The results showed that the optimal coating concentration of rabbit polyclonal antibody and the optimal dilution ratio of mouse polyclonal antibody were 1∶1 600,the optimal blocking condition was 3％skimmed milk powder for 1 h,the optimal dilution ratio of enzyme-labeled secondary antibody was 1∶4 000,the incubation time of enzyme-labeled secondary antibody was 1 h,and the color development time was 15 min.The core antigen component BCSP31 protein of Brucella chimeric virus-like particle vac-cine(BCSP31-cVLPS)was quantitatively detected under the above optimal reaction conditions,and its sensitivity,specificity and repeatability were evaluated.The results showed that the BCSP31 content in 1 μL Brucella chimeric virus-like particles accounted for 14.187％of the total protein content.After diluting of Brucella chimeric virus-like particles 640 times,the P/N value was still greater than 2.1,and there was no cross-reaction with other virus-like particles such as FAdV,IB-DV,NDV and AIV.Specificity,inter-and intra-batch coefficients of variation were all below 10％,and repeatability was good.The establishment of this method lays a foundation for promoting the establishment of quality control standards and commercialization of Brucella chimeric virus-like particle vaccine.
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