The Development Of A Novel Monoclone For Vitamin D Binding Protein

Objective:Vitamin D is an important fat-soluble vitamin in the human body,which is mainly synthesized by skin under ultraviolet irradiation,circulates to blood,binds with vitamin D binding protein(DBP),and forms the compounds of vitamin D--DBP which are the main forms of existence in our body.Then the compounds transport to liver and kidney,are hydroxylated twice to biological active 1,25(OH)2D,combine with VDR of each target tissue to exert its biological function.Vitamin D regulates the absorption of calcium and phosphate in the body,maintains the levels of calcium and phosphate in blood,and promotes human teeth and bones normal development.And the level of vitamin D in body is correlated with a lot of systems’ diseases,sufficient vitamin D can prevent some diseases.ELISA is a specific binding between antigen and antibody,which is used to detect special antibodies.Antigen or antibody which has immune activity combines on solid phase,with enzyme color reaction,it can display specific antigen or antibody exists or not,and according to the color depth to do quantitative analysis.ELISA is a method that can rapidly determine the concentration of antibody or antigen.So using the principle of ELISA can develop a new antibody of vitamin D binding protein.Methods:This experiment use two kinds of vitamin D binding protein peptides to inject two groups mice respectively to immune,control group inject normal saline,take the spleens of mice which have high immunity after immunization three times comparing with control among three months.Then the spleen fuses with SP2 cells,and selects with HAT,HT and complete medium in turns in 96 well plates.Using indirect ELISA to screen the cells of successful fusion,called hybridoma,and to clone the hybridomas having high expression of antibody,further to test the antibody produced by hybridomas with sandwich method and to purify,produce the antibody in accordance with regulations.Results:In the two types of hybridoma screening,the color changes;and to test the antibody produced by hybridomas,the color also changes correspondingly.Conclusion:Screening the fused cells,it is successful to fuse for spleen and SP2 cells when the color of fused cells deeper than control;also to detect antibody,if the color of hybridoma is deeper than control,it is suggested that the hybridomas have a positive expression,the clones could be successful to produce antibodies,can be combined with vitamin D binding protein specific.