| The myocardial injury caused by Acute myocardial infarction (AMI), Acute coronary syndrome (ACS), Unstable angina pectoris (UAP) et al, and other cardiovascular disease is a major killer in our country as well as in the developed world. One of most critical issue in acute cardiac event is to the correct and promptdiagnosis for proper treatment. However, due to the often asymptomatic features of the diseases, the diagnosis for early myocardial injury is difficult. Though major efforts have been taken by medical researchers to find meaningful biochemical and/or molecular markers and their corresponding diagnostic techniques, there are still needs for biomarkers and diagnostic methods that meet the clinical purposes more satisfactory.Based on numerous research publications and structural analysis, we elected myosin as a molecular marker for myocardial injury. In order to obtain diagnostic monoclonal antibodies specifically against cardiac myosin,and develop myocardial injury diagnosis methords, three major tasks are undertaken:1) Development of porcine cardiac myosin monoclonal antibodies Our preliminary studies showed that cardiac myosin may be more sensitive and specific than electrocardiogram and other _,serum enzymes and myoglobin in the diagnosis of myocardial infarction. When cardiac muscle is damaged or cardiac cells are in a low pH environment, cardiac myosin is cleaved and its light chains (CMLC) and heavy chains (CMHC) are released into the interstitial space and then blood. The CMLC release seems to appear early and last a long time and the CMHC are specific, so myosin may be used in both early diagnosis of AMI and late stage monitoring. The testing of myosin could be one of the most specific biochemical markers in the diagnosis of myocardial infarction. Since the amino acid sequence of cardiac myosin heavy chain between porcine and human's shares more than 99% homology, and since it is difficult to obtain human cardiac myosin samples as antigen, we used cardiac myosin purified from porcine cardiac muscle to immunize mice for antibody production. In seven hybridizations with indirect ELISA method, the 14 strains of hybridoma selected showed stable secretion of anti-porcine cardiac myosin McAbs with high tilers from 5 wells of hybridoma from over 3,000 wells. It is shown in preliminary experiments that the obtained McAbs posses thecharacteristics of high affinity and sensitivity, thus providing a good basis for the establishment of diagnostic methods.2) Development of double-antibody-sandwish EL1SA assay for detecting human cardiac myosin in plasma and it' s primary application Anti-cardiac-myosin monoclonal antibodies were coated to ELISA plates, and the Horseradish Phosphotase (HRP) labeled rabbit anti-cardiac-myosin polypeptide antibody as the second antibody to establish a sandwich ELISA to detect cardiac myosin in serum quantitatively and qualitatively. The minimal detectable cardiac myosin in sera was 0.3 ng/ml with our antibodies. And the standard curve shows good linearity from 1.0 to 20 ng/ml of cardiac myosin. We employed the ELISA method to determine the cardiac myosin levels in serum samples of thirty AMI patients with confirmed clinical diagnosis by local hospitals and clinics. When comparing with other methods, the ELISA showed a corresponding rate of 84.8% and has good reproducibility and stability.3) Development of a colloidal gold immunochromatographk assay for determination of human cardiac myosin in serum and it' s primary application In this study, another technique, lateral flow immunoassay, was employed to assess the cardiac myosin marker. Gold labeled anti-myosin polypeptide antibody of good stability and immune reactivity was previously prepared by polypeptide immunolization of rabbits and affinity column purified. The labeled polypeptide antibody was fixed on cellul&se membrane to act as a color source. Another antibody oranti-cardiac-myosin monoclonal antibody was linked to nitro cellulose membrane as capture antibody. The sensitivity o... |
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