| Acute myocardial infarction (AMI) is a killer to human health as the most serious type of acute coronary syndrome (ACS), and can cause sudden cardiac death. AMI can be divided into ST-segment elevation myocardial infarction (STEMI) and non-ST segment elevation myocardial infarction (NSTEMI) according to the changes of ECG. STEMI has the characteristic changes of ECG, can be found and treated early, the prognosis is better. NSTEMI dosen’t have the characteristic changes of ECG, which is particularly important to detect cardiac markers for early diagnosis and treatment of NSTEMI in this case. Cardiac troponin I (cTnI) has high sensitivity and specificity to myocardial injury, which has become the first choice in cardiac markers for the diagnosis of AMI, and has been considered as a " gold standard " for the evaluation of myocardial necrosis and the diagnosis of AMI.TnI is one of the three subunits of major myocardial contractile proteins, which is the inhibition subunit of myofibrillar ATP, and is comprised of fast skeletal isoform (fTnI), slow skeletal muscle isoform (sTnI) and cardiac isoform (cTnI). cTnI has about40%heterologous to other subtypes, which extends a sequence comprised of31amino acids in the amino terminal, and can improve the specificity and accuracy of diagnosis of AMI with this characteristic. cTnI experiences the improvement of detection methods and the study of clinical application from the conventional method to highly sensitive method, high-sensitity cTnI (hs-cTnI) has been widely recognized and applied in clinical practice. Based on luminol oxygen channel immunoassay (LOCI), we establish a new quantitative method for cTnI detection, which has many advantages such as high sensitivity, homogeneous, no washing, rapid, simple and so on.Objective:Based on luminol oxygen channel immunoassay (LOCI), in AlphaScreen platform, we initially establish a homogeneous chemiluminescent immunoassay system for cardiac troponin I (cTnI) detection and make a evaluation of methodology and clinic.Methods: Mouse monoclonal antibody for cTnI and rabbit polyclonal antibody for cTnI coated receptor microsbeads and biotin respectively, composed of two antibodies, chessboard titration is used to determine the best combination, which is used to determine the optimal concentration of the receptor microbeads and the biotinylated antibody; two phase reaction time, reagent and sample order, the proportion of reagent and sample,and buffer of the detection system were optimized. Based on optimizing the four parameter fitting equation of standard curve, to detect the clinical specimens of unknown concentration and get the emitting signal, according to the curve we can obtain the corresponding cTnI concentration and make methodology and clinical evaluation. Study by ELISA Calc software for statistical analysis.Results:This cTnI detection system consists of three parts:rabbit polyclonal antibody for cTnI coated receptor microbeads, mouse monoclonal antibody for cTnl coated biotin and avidin coated donor microbeads, the method is rapid and sentitive, the detection time is17.5min; analysis sensitivity of0.045ng/mL, functional sensitivity of0.053ng/mL; Recovery is104.97%-108.12%; within-run precision is3.88%-5.53%, between-run precision between7.60%-8.75%; the interference rate of hemolysis, jaundice, and lipidemia<10%; This paper established methods cTnI reference limit1.05ng/mL;40clinical specimen in the testing results correlate well with direct chemiluminescence detection assay results, r2=0.979; the chi square test and consistency analysis indicate that the system and direct chemiluminescence detection reagents are no statistical difference.Conclusion:This system can be used for quantitative detection of serum cTnI, and with homogeneous, no washing, strong sensitivity, high accuracy, good repeatability, easy operation etc, and further research is expected to promote the clinical application. |
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