| Rotavirus is the most common cause of the child non-bacterial diarrhea in the worldwide,.Every year there are approximately 800,000 children die of the rotavirus infection.Therefore,the safe and effective vaccine is the principal means to prevent RV infection.Rotavirus,a member of the family Reoviridae,is divided into seven groups(A-G) according to the antigenicity of the structure protein VP6.RV vaccines at present aim directly at group A rotaviruses,the major cause of severe dehydrating gastroenteritis among the infants and children.RV diarrhea has already become a severe public health problem and induced heavy disease burden and economic burden to society.There is no specifics to RV diarrhea now.And the improvement of water quality and public health condition can not cut down the attack rate of RV diarrhea obviously.Therefore,to develop RV vaccine in high performance,safety and lower price is the only approach to prevent and control RV infection.WHO has been thinking highly of the research and development of RV vaccine.The RV vaccines utilized clinically are all attenuated live vaccine for oral use,which has gained fine immunization and protection effect when taken by susceptible population.However, because of the danger of actvistic heredity and potential severe side effect,the attenuated live vaccine is not considered as the main aspect of the research of RV vaccine yet.Inactivated vaccine is becoming a hot spot in the research of RV vaccine gradually in advantage of its safety and stability.The RV antigen qualitative and quantitative detecting has the vital significance to the vaccine research.How to determine RV antigen quantity is a very important link and technology in the RV vaccine research specially RV inactivated vaccine.A precise RV antigen quantity detection system is very essential to the preparation of vaccines,the immunity dose determination,the vaccine allocated proportion,the immunization schedule's determination,the immunity effect evaluation and so on.Having effectively immunogenic antigen is one of the most important conditions to make good inactivated vaccines.How to accurately detect the antigen quantity after inactivation is crucial to research and preparation of inactivated vaccine. This research has done a series of related work including massive RV antigen preparation,RV culture solution concentration,purification,animals immunity test, specific antiserum separation and identification as well as rotavirus antigen quantitative ELISA system exploration.The neutralization test results indicate the titer of antibody originated immune animal reaches to 1:256.We use the above antibody and commercial RV McAb to carried out multifactorial RV antigen ELISA detecting.In this experiment,using antibody pairs were analyzed to choose the best coating antibody and detecting antibody.The experimental result indicated that McAb must be taken as coating antibody,PcAb be taken as detecting antibody.the detection reproducibility as well as the sensitivity was superior to PcAb as coating antibody and McAb as detecting coating.In this research,we discover that overtop or lower coating antibody,detecting antibody and enzyme union will have greatly influence on the test result.In our experiment,the coating antibody,the detecting antibody,the enzyme union best concentration respectively is 1:1000,1:200,1:5000.and we also optimizes the diaper buffer solution as well as other ELISA operating conditions.Enhances response specificity as well as stability.The research has preliminarily established RV antigen quantity ELISA detecting method,we use the different antibody level to detect antibody,different origin anti-RV McAb,different orgin enzyme union as well as different RV strain,whether have the difference to make the further research. |
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