Study And Standardization On Methods Of Total Protein Content Test Of Foot-and-mouth Disease Inactivated Vaccine

In actual production process, vaccine purity which affects differentiation vaccinated from infectedanimals tests (DIVA tests),as well as relates to side-reactions of vaccination, is one of the key factor inquality control of FMD inactivated vaccine.Improving productive process, increasing vaccine effectiveantigen content(146S) and reducing protein content such as non structure protein of FMDV in order toproduce high quality FMD inactivated vaccine was the domestic reformation of the productivetechnology of FMD inactivated vaccine. Because the protein quantitation of FMD inactivated vaccinecould indirectly reflect the purity of vaccine, so it is quite necessary to establish an accurate, efficientand simple method for protein quantitation of FMD inactivated vaccine.In this study, three protein quantitation assays including Kjeldahl nitrogen method, ModifiedLowry protein assay and Coomassie Plus assay were compared, and one method which was accurate,efficient and simple was screened to quantitate protien content of FMD inactivated vaccine. In thismethod,the antigen in FMD inactivated vaccine was replaced by standard albumin which the qualityconcentration was known to simulate the inactivated antigen in FMD inactivated vaccine, in order tooptimize the operating procedures of the method which have been selected.The BSA was diluted fordifferent concentrations to verify the sensitivity of this method, and the repeatability and reproductivitywere also verified by quantitating protein content of FMD inactivated vaccines from differentmanufacturers by optimized operating procedures of this method.Results indicated that Modified Lowry protein assay could perform protein quantitation more truly,and it was simple, efficient, and low consumption compared to the Kjeldahl method. The optimaloperating procedures of the method was that the vaccines were dealt with actone first,and then theprotein that precipitated by actone was suspended and quantified. In this study,the practicalmeasurement range of the Modified Lowry protein assay kit was from50μg/mL to2500μg/mL. Therepeatability and the reproductivity of the optimal operating procedures of the method were very good.The measurement results of37batches vaccines from different manufacturers indicated that proteincontent of different vaccines from different manufacturers was significantly different, but about94.6%of the protein content of vaccines was in the range of50μg/mL to2500μg/mL,and there was nomanagement such as dilution or concentration were needed in measuring process.The established operating process of Modified Lowry protein assay in this study could be appliedto protein quantitation of FMD inactivated vaccine.It provided experimental basis for the selection andstandardization of protein quantitation methods for FMD inactivated vaccine. And this methods hasbeen recommended to quantitate protein content of FMD inactivated vaccine by the Ministry ofagriculture.