Purification And Characterization Of Troponin T From Bovine Cardiac Muscle And Production Of Its Monclonal Antibodies

The troponin T of bovine cardiac muscle (cTnT) was purified approximately 280 fold with 14% yield from LiCL extraction, ammonium sulfate salting out and ion-exchange chromatography. In TSK G2000 SW HPLC chromatography, cTnT showed one sharp peak with Mr of 37KD. The SDS-PAGE electrophoretic data on subunit composition revealed that the troponin complex is composed of three subunits with Mr of 17KD, 24KD, 37KD respectively. The pi of cTnT is 7.2. Amino acid composition analysis of cTnT revealed that Lys, Glu, Asp, Ala were the major components.The purified cTnT was used to immunize the BALB/C mice. Four hybridoma cell lines, which secreted monoclonal antibodies (McAbs) consistently, were obtained through cell fusion, screening and cloning. Isotype of these four McAbs are all IgG2b . The litres of the ascitic fluids of four McAbs measured by ELISA analysis were 1/100000 , while supernatant were 1/100. Their specific reaction is to cTnT only, not to skeletal troponin T(sTnT), cardiac troponin I(cTnl), human IgG, bovine serum albumin by using ELISA and immunoblotting.The production of specific McAbs against cTnT provides an important means in the further studies of relationship between cTnT and cardiac injury.