Optimization Of High-sensitivity CTnI Immunochromatography And Its Application In Dry Biochemical Immunoassay System

At present,the gold standard for clinical diagnosis of ACS syndrome are the concentrations of I subunit(cTnI)and T subunit(c Tn T)from cardiac troponin(c Tn)in various international clinical guidelines.Negative prediction accuracy for myocardial infarction can reach 99% with high-sensitivity cTnI(hs-cTnI)detection reagent.The missed detection rate of ACS can be significantly reduced combined with other clinical auxiliary diagnosis methods.However,the current clinical used reagents have weak points including slow detection speed and low sensitivity.It's imperative to develop a more accurate and faster cTnI detection reagent to screen out ACS cases,prescribe the right medicine,and alleviate the crowdedness of the emergency department.High concentration cholesterol is one of the important causes of myocardial infarction by formation of atherosclerosis in the heart blood vessels,leading to myocardial ischemia and myocardial infarction.Measuring of cholesterol related biomarkers and cTnI concentration at the same time can provide more information on patients,which resulting better treatment plan and clinical guidance.However,measurements of cholesterol related biomarkers are carried out on large-scale biochemical instruments,beyond the chest pain center's easily and quickly access.Lateral flow immunochromatography technology is a classic in vitro diagnostic technology platform,which can be applied to the detection of various markers.It has advantage in quick measurement,and has low costs for production,storage,and transportation,resulting of widely clinical using.However,lateral flow immunochromatographic reagents have the problems of insufficient sensitivity,low accuracy,and batch-to-batch differences in products.In this thesis,a new dry-type biochemical immunoassay lateral flow chromatography system,with high-sensitive and high-precise cardiac troponin I detection,was established with improvement on sensitivity and accuracy of lateral flow chromatography system.The performance and scalability of the POCT platform have been significantly elevated.The main contents of this thesis are shown as follows.(1)cTnI antibodies with high binding affinity and comprehensive response were screened,based on enhanced chemiluminescence(ECL)Western blotting technology with native state of cTnI.The selected antibodies pair was confirmed through the verification of clinical samples to eliminate the missed detection and insufficient sensitivity of cTnI.(2)The components of the lateral flow chromatography system were studied to improve the system accuracy.The precision of the reagents is controlled within 10%-15% by optimizing the sample pad treatment solution formula;the precision is controlled at about 6% by adjusting the mixing method of the fluorescent microspheres and the sample.The cTnI concentration measurement time is less than 10 minutes,and the precision is controlled within 5% with a set of microfluidic chips.(3)Internal calibration system was introduced to improve the detection precision of cTnI concentration.BSA calibration system was selected after screening.The optimized reaction conditions of BSA calibration system were determined as follows: monoclonal antibody mono of cTnI and 1mg/ml BSA were mixed and coated together;goat polyclonal antibody mixed with Cy5 at molar ratio of 1:2.The detection precision of cTnI can reach 3-5% with BSA calibration system and meet the clinical requirements for accurate quantitative testing.(4)A combined quantitative reagent based dry biochemical immunoassay system was designed for eight biomarkers of CK-MB,Myo,cTnI,cholesterol,triglycerides,high-density lipoprotein cholesterol,low-density lipoprotein cholesterol and uric acid.Firstly,the standard curves were fitted to obtain the measurement ranges and LOD of the reagent required by clinical diagnosis.The repeatability was measured and specificity was determined with all the biomarkers to get rid of cross-interference among these biomarkers.Finally,this reagent was tested on clinical samples to get comparison results.More clinical results can be obtained in shorter time with dry biochemical immunoassay system,showing great potential in clinical diagnosis.