| Human cardiac troponin I (cTnl) was an important biochemical marker for myocardial injury. However, until recently, the detection of cardiac troponin I had various limitations because the difference of the clearance rate and the degradation rate of cTnI peptides, the antibody against the different antigenic epitope, the various affinity of antibody, using diverse reference standards and so on. According to research, cTnI results varied 100-fold among different assays. It was necessary to standardize the cTnI protein and difference assays of cTnI measurement in clinical. On the other hand, china does not independently have a cTnI diagnostic kit. The clinical cTnI diagnostic kits are from abroad or domestic assembly by imported raw materials. In this way, it not only increasing the test cost but also increasing the economic burden of patients.Those factors are limiting cTnI, the more sensitivity and more specificity of biochemical maker, applied in the clinical.Anti-cTnI hybridoma cell lines were obtained by recombinant human full-length cTnI as antigen. The purpose of this research was to obtain the sufficient cTnI antibody with the higher specificity and higher titer, and to develop the colloidal gold immunochromatographic assay for the rapid detection of hcTnI.The monoclonal antibody is obtained by induced ascite in vivo, preliminarily purified by caprylic acid-ammonium sulfate precipitation and further purified by Protein A Sepharose CL-4B affinity chromatography. Polyclonal antibodies were made by immunizing New Zealand rabbit and purified by caprylic acid-ammonium sulfate precipitation.Threre are several methods to preparation of colloidal gold and the main principle is chloroauric acid aggregated into a specific size of gold particles under the reducing agent. In this reaserch, we successfully established a colloidal gold immunochromatographic strip by using immunity chromatography technology. First, the mouse anti-cTnI monoclonal antibody was conjugated with colloidal gold particles prepared by the citrate method. Second, the rabbit polyclonal antibody was immobilized on the nitrocellulose membrane to capture the colloidal gold-mouse anti-cTnI monoclonal-cTnI complex. Last, the goat-anti-mouse IgG was also immobilized on nitrocellulose membrane to combine labeled antibodies as the control line.The result of this strip was compared with the colloidal gold kit of alpha science company. We successfully obtained the cTnI colloidal gold immunochromatographic strips by the optimization of conditions. The detection range of colloidal gold immunochromatographic strip is 5ng/ml to 1μg/ml; Sensitivity:5ng/ml; Specificity:the test is no cross-reaction with myoglobin, creatine kinase-MB, cTnT and cTnC; compared with the colloidal gold kit of alfa scientific designs inc:the positive coincidence rate was 96.4%and the negative coincidence rate was 100%.Conclusion:successfully established the colloidal gold immunochromatographic assay exhibiting high sensitivity and specificity to detect the cTnI of samples, and this assay is rapid, economical, and simple, without the requirement of complicated equipment. It can be widely used for the early diagnosis of AMI and scientific research. |
PDF Full Text Request