Establishment And Clinical Application Of Highly Sensitive Detection Methods For Total CTnⅠ,Full-length CTnⅠ,cTnⅠ-C Complex

Cardiac troponin Ⅰ(cTnⅠ)is a serological marker for diagnosing myocardial injury.It can be used to evaluate myocardial injury-related diseases such as acute myocardial infarction and acute coronary syndrome.After myocardial injury occurs,cTnⅠ will be released into the blood and gradually degraded in the blood.Its main form is cTnⅠ-C complex.Serum cTnⅠ contains both undegraded full-length cTnⅠ and degraded cTnⅠ fragments.Existing methods generally detect the total serum cTnⅠ content of patients without considering their degradation.At present,there are few reports on the clinical diagnosis value of serum cTnⅠ degradation to myocardial injury.The purpose of this study was to establish a combined detection method for full-length cTnⅠ,total cTnⅠ,and cTnⅠ-C complex in serum.The total cTnⅠ,cTnⅠ-C complex and full-length cTnⅠ/total cTnⅠ ratio were used to determine the degree of myocardial injury and Duration of onset.In this study,a monoclonal antibody C30 targeting the stable region of cTnⅠ was uniformly coated as a capture antibody,and three different monoclonal antibodies were labeled with rare earth ions to establish the corresponding time-resolved fluorescent immunoassay(TRFIA).Monoclonal antibody L83 targeting another stable region of cTnⅠ was used as a labeled antibody for total cTnⅠ detection,monoclonal antibody L190 targeting the C-terminus of the cTnⅠ molecule was used as a labeled antibody for full-length cTnⅠ detection,monoclonal antibody antibody L20C6 targeting the cTnⅠ-C complex is used as a labeled antibody to detect the cTnⅠ-C complex.Through the optimization of coating concentration,dilution of labeled antibody,reaction time and other conditions,three cTnⅠ-TRFIA methods were established:Total-cTnⅠ-TRFIA(to detect total cTnⅠ),Full-cTnⅠ-TRFIA(to detect full-length cTnⅠ),CTnI-C-TRFIA method(to detect cTnⅠ-C complex).The three cTnⅠ-TRFIA methods established were evaluated methodologically,and their sensitivity,precision,specificity and accuracy were analyzed.After the three cTnⅠ-TRFIA methods were evaluated,the total serum cTnⅠ,full-length cTnⅠ,cTnl-C complex concentrations of the sample serum were measured,and the correlation between the detection results of the three methods and the detection results of the clinical cTnⅠ chemiluminescence immunoassay was analyzed.By continuously detecting the concentration of serum full-length cTnⅠ,total cTnⅠ,cTnⅠ-C complex at different times in three patients with myocardial injury,calculate the proportion of full-length cTnⅠ at different times(full-length cTnⅠ/total cTnⅠ),combined with clinical observation results to compare the value of serum cTnⅠ concentration(total cTnⅠ concentration,full-length cTnⅠ concentration,cTnⅠ-C complex concentration)and the proportion of full-length cTnⅠ and clinical cTnⅠ detection value measured by the three methods in judging the duration and prognosis of patients.The research results show that the sensitivity of Total-cTnⅠ-TRFIA is 0.03 ng/mL,the intra-batch coefficient of variation(CV)is 4.78%,the inter-batch CV is 7.97%,the detection range is 0.03-40ng/mL,and the recovery rate is 106.62%;The sensitivity of Full-cTnⅠ-TRFIA is 0.007ng/mL,the intra-batch CV is 3.59%,the inter-batch CV is 8.06%,the detection range is 0.007-40ng/mL,the recovery rate is 102.23%;the sensitivity of cTnⅠ-C-TRFIA is 0.011ng/mL,intra-batch CV is 4.72%,inter-batch CV is 8.83%,detection range is 0.011-40ng/mL,recovery rate is 92.15%.Troponin T(cTnT)did not cross-react with the three methods.Therefore,the sensitivity,precision,and recovery rate of the three cTnⅠ-TRFIA tests established all meet the requirements of diagnostic reagents.In terms of correlation with clinical cTnⅠ-CLIA detection methods:Total-cTnⅠ-TRFIA method has the highest correlation with clinical detection methods(R2=0.9225),and Full-cTnⅠ-TRFIA method has the lowest correlation with clinical detection methods(R2=0.6238),The correlation between cTnⅠ-C-TRFIA method and clinical detection method is between them(R2=0.8868).In the detection of cTnⅠ concentration in serum samples:the average concentration of cTnⅠ measured by Total-cTnⅠ-TRFIA method is the highest,its concentration is 5.59 ng/mL;the average concentration of cTnⅠ measured by cTnⅠ-C-TRFIA method is the second,and its concentration It was 4.01 ng/mL;the average concentration of cTnⅠ measured by Full-cTnⅠ-TRFIA method was the lowest,and its concentration was 2.12 ng/mL.The average concentration of cTnⅠ measured by the clinical cTnⅠ-CLIA assay is 4.38 ng/mL,which is between the results of the Total-cTnⅠ-TRFIA method and the cTnⅠ-C-TRFIA method.Among the 77 positive sera detected by all methods,the cut-off value of clinical cTnⅠ-CLIA detection(0.11 ng/mL)was used as the criterion for diagnosing cTnⅠ abnormality:the detection rate of clinical cTnⅠ-CLIA was 100%(77 cases),The detection rate of Total-cTnⅠ-TRFIA method was 100%(77 cases),the detection rate of Full-cTnⅠ-TRFIA method was 90.9%(70 cases),and the detection rate of cTnⅠ-C-TRFIA method was 98.7%(76 cases).In the analysis of the total cTnⅠ,full-length cTnl,cTnⅠ-C complex and the proportion of full-length cTnⅠ on the serum of three consecutively tested patients:the concentration of total cTnⅠ and cTnl-C complex can well reflect the patient’s myocardial injury Degree;the proportion of full-length cTnⅠ can better indicate the time of onset,and the prognosis is earlier than the concentration change of the total cTnⅠ and cTnⅠ-C complex.The combined detection of serum full-length cTnⅠ and total cTnⅠ and calculation of the proportion of full-length cTnⅠ has a better prospect for clinical diagnosis and treatment,and is helpful for the diagnosis and therapeutic effect evaluation of myocardial injury.