The Development of Elisa Kit Special to Human Tissue Kallirein
|School||Huazhong University of Science and Technology|
|Keywords||Tissue kallikrein Fusion protein Separation and Purification monoclonal antibody polyclonal antibody|
Studies have shown that Kallikrein Kinin/bradykinin system KKS play a role incardiovascular disease,cerebrovascular diseases, diabetes along with its complications,cancer and so on. Also, researches confirmed that the relationship between the quantity ofkallikrein in the blood and ischemic stroke suggest that kallikrein can be used as animportant vitro diagnostic indicators for stroke. In view of some research conclusion andthe study results that we have done, we decide to develop the Elisa kit special to humantissue kallirein.Object To develop a quantity detected ELISA kit special to kallikrein, lay thefoundation for the diagnosis of stroke, coronary heart disease, diabetes and so on.Method (1)Amplified the KLK1target gene with specific primers, and applied positiveobject of the recombinant plasmid HK/4T1to transforming into E. coli DH5α and thenprokaryotic expression and purification, and further optimize the conditions.(2) Preparationof monoclonal antibodies:inject mice with hybridoma cell, inducing ascites which producemonoclonal antibody.the titer of the monoclonal antibody could be1:1×10^5. Highpurity, high titer antibodies purified with AKTA system can be used for the ELISA test.(3)Preparation of polyclonal antibody for KLK1reagent packer orifice, in order to improve theaccuracy of the detection kit; Do ELISA assay with the direct method in non coatedmicrotiter plates at the same time.Result (1) SDS PAGE electrophoresis with the fusion proteins induced by IPTG, resultsshowed that a stain of additional protein bands was at the department of55KD. Thewestern blottings indicate that this recombinant protein present antigen of KK.(2)campared with the cell culture medium supernatant, the monoclonal antibody produced bymouse ascites tend to be higher purity and titer.(3) OD values of wells coated withpolyclonal antibody present with the downward trend following reduction of the concentration of antigen, but linear relationship is poor; linear relationship of the OD valuein the direct method appears well under0.75ug/ml.Conclusion (1)we have successfuly cloned and expressed the human tissue kallikrein gene.Under optimum conditions, the major in the initial separation of the product is the targetedprotein.(2)we have initially prepared monoclonal antibody and ELISA kit specified totissue kallikrein.(3)the next object is mainly concentrated in the detection of the enzymecontent of human tissue kallikrein,along with how to industrializate and improve theaccuracy and repeatability of the test method.