Development Of Cardiac Troponin ? Rapid Quantitative Analysis Test Kit Based On Time-resolved Microsphere Immunoassay

Cardiac Troponin I(cTnI)is a highly correlated marker for the diagnosis of Myocardial Injury(MI).For the diagnosis of MI,cTnI has the advantage of high sensitivity,good specificity and long-lasting,which makes it become the best marker to analysis the risk of MI in patients who have the Acute Coronary artery Syndrome(ACS),cTnI already became the gold standard for the diagnosis of Acute Myocardial Infarction(AMI),by replacing Creatine Kinase isoenzyme MB(CK-MB).Along with the deeper research on MI and cTnI,the sensitivity and real-time performance of the cTnI detection are getting higher requirement in clinic.The paper aims at researching on the method of using time-resolved microspheres to detect cardiac troponin I with the rapid quantitative immunoassay.The method combined both time-resolved Microsphere Assay and immunochromatography,which has the characteristics of simplicity and small amount of specimens needed by chromatography;it also has convenience of point of care test(POCT)and the characteristics of time-resolved Microsphere method which is low noise and high sensitivity.Based on that,it can provide a rapid,simple and accurate detection,and save time to improve the survival rate of patients.This assay focused on four aspects of research.Material selection included antibody,time-resolved microsphere and nitrate cellulose membrane.It was evaluated with three parameters including sensitivity,specificity and stability.This method were using three monoclonal antibodies named 7B9?560 and 20C6.7B9 and 560 which were coated onthe nitrate cellulose membrane 95 with 0.5mg/mL,separately.And 20C6 was used as detected antibody with 0.4mg/mL labeled with 300 nm time-resolved microspheres,leading to a high sensitivity.By exploring the labeling technology of time-resolved microspheres,for obtaining higher protein coupling rate and sensitivity,The activation used 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride(EDC)and N-Hydroxysuccinimide(NHS)as the cross-linking agents,under the condition of borate buffered solution(BBS)with pH 8.0.Then the reaction was performed with protein in 2-(N-Morpholino)ethanesulfonic acid hydrate solution(MES)with pH 6.0.In the aspect of reagent technology research,the traceability chain was formed with Beckman AccuTnI Enhanced a Hytest cTnI calibration product,which was relatively consistent with the consensus of international standardization.Vacuum drying of conjugate(microsphere and antibody)had improvement of the sensitivity and precision of test.And the test time of the assay could also be shorten to 10 minutes.With the systematic evaluation of Pilot Product Performance,the Coefficient of variation could be controlled within 10 %,and the minimum detection limit is 0.03 ng/mL,and the functional sensitivity is fixed for 0.08 ng/mL,as they give expression to excellent sensitivity.It had a strong anti-interference ability with the lower cross-reaction rate less than 0.001 %.The correlation coefficient R with the Beckman AccuTnI Enhanced reagent kits was equal to 0.978,which has good consistency and meets the needs of clinical testing.