The Preparation Of Monoclonal Antibodies Against Rotavirus

Rotavirus is an important factor in causing severe diarrhea around world children,caused by Rotavirus infection is not only seriously affect infants and young children’s health and quality of life, and bring huge economic burdens on families and society. It shows that rotavirus infection has become an urgent major public health problem worldwide. There is no cure for the treatment of rotavirus infection currently, therefore,the study of a rapid and accurate method for the early detection of rotavirus is of far-reaching significance for the prevention and treatment of the virus. Monoclonal antibodies have the characteristics of high specificity and high sensitivity. If it is applied to the detection of rotavirus, rotavirus infection can be timely diagnosis. This research is committed to the preparation of monoclonal antibodies against rotavirus, to provide the fundamental basis for the establishment of an effective rotavirus diagnostic test.The first chapter introduced the basics associated with rotavirus, including the shape and biological characteristics of the virus, in addition, common methods of detection of rotavirus in recent years are reviewed, and summarized the factors during preparation of monoclonal antibodies based on the experimental work experience.Duing to the longer experimental period of the preparation of monoclonal antibodies,and the more experimental procedures, if there is something wrong, it will directly affect the final results, so enumerated considerations in all aspects of the experiment,in order to avoid to arising more problems during the research.The second chapter examed experimental conditions investigated all aspects,including the dilution times of antigen, serum dilution, the selection of blocking solution, the determination of blocking conditions, the coating time of enzyme-labeled secondary antibody and the color time of substrate solution. Establishing an ELISA assay for rotavirus protein, it allowed for the detection of related antibodies in the preparation of monoclonal antibodies easily.The third chapter introduced the process of monoclonal hybridomas obtained by cell fusion, including the immunize mice, cell fusion, screening of positive hybridomas and the acquisition of monoclonal hybridomas. This research obtained five stable hybridoma cell lines which are capable of secreting monoclonal antibodies finally:1B3-A2, 1B3-C3, 2B4-A9, 3D19-C2, and 4E22-B10.The fourth chapter stated the purification of antibodies after producing a number of monoclonal antibodies and further examined the concentration, specificity and biological characteristics of the antibodies after purification. Adopting monoclonal antibodies superposition experiment,it can be obtained that:apart from 1B3-A2 and4E22-B10 two hybrisoma cells which have the potential of secreting monoclonal antibodies against the same epitope possiblely, 1B3-C3,2B4-A9,3D19-C2 which can secret monoclonal antibodies against different epitopes.