Optimization And Purification Of Human Cardiac Troponin I(cTnI) In Lab And Preparation Of Antibodies Against CTnI

Human cardiac troponin I (cTnl) was an important biochemical marker for myocardial injury. However, until recently, cardiac troponin testing had various limitations because the antibody against different antigenic epitope, varies affinity of antibody, distinct clearance rate of cTnI peptides, and using diverse calibrators. According to research, cTnI results varied 100-fold among assays. It was necessary to standardize difference assays of cTnI measurement in clinical. On the other hand, limiting resource of cTnI contributed to search genetic method to supply with sufficiency cTnI. By constructing genetic bacteria expressing cTnI provided us unlimited cTnI proteins.The purpose of this research was optimizing expression of cTnI in Escherichia coli (E.coli) which had been constructed. At same time, cTnI expressed effectively. Then, cTnI were purified from E.coli and used as antigen immunized BALB/c mice and New Zealand big ear rabbit to prepare monoclonal antibody and polyclonal antibody. Which maybe as calibrators to harmony the difference assays of cTnI measurement in clinical.The factors influenced expression of cTnI in E.coli include:temperature, pH, shaking speed, concentration of IPTG, induction time and construction of medium. The conventional methods of optimization involved changing variable-to-variable while maintaining all others at affixed level did not yield reliable results because interactions between different components were neglected. Besides, they were laborious, time-consuming, and impractical. Plackett-Burman design (PBD) which ignored interactions among factors had been applied to minimize the number of factors. The response surface method (RSM) was further adopted to determine the relationships between variables and responses. Those two steps method did not only reduce laborious but also most optimizing variables searched.Ammonium sulfate deposition and ion exchange chromatography procedures were used to purify cTnI from E.coli. SDS-PAGE show that purified cTnI contained one band. cTnI could be used to immune animals as an antigen to produce monoclonal antibodies with high affinity and specificity. To prepare cTnI monoclonal antibodies, the spleen cells of immunized BALB/c mouse fused with myeloma cells(Sp2/0). Polyclonal antibodies were made by immunizing New Zealand rabbit.The characters of cTnI were identified by indirect ELISA and Western-blot.5 hybridomas, named C5B2, C5B3, C5B4, C5B1 and B1A6, could secret cTnI McAb. B1A6 was selected to identity. It belonged to IgG3 and IgG2b subtypes and had high titer and specificity. The monoclonal antibodies and polyclonal antibodies against McAb were prepared and identified. The Koff of monoclonal antibody is 1.08x10-9mol/L. The titer of polyclonal antibodies was 1:5000.Conclusion, we obtained pure cTnI after optimization and purification. Then, we produced affinity monoclonal antibody and polyclonal antibody against to cTnI.