Study On Accurate Quantitative Method Of Immunoactivity Concentrations Of Two Myocardial Injury Biomarkers

Cardiovascular disease is a common clinical disease in China,and patients’conditions tend to get critical.The early diagnosis of cardiovascular disease needs to be combined with clinical symptoms,electrocardiogram,and detection of myocardial injury biomarkers.Biomarker detection is an important method for clinical non-invasive diagnosis of cardiovascular diseases.Myoglobin and transferrin are early diagnostic biomarkers and acute phase resaction markers of myocardial injury.The accurate quantification of hMYO and hTRF is helpful for the early diagnosis and judgement of state of patients with cardiovascular disease.Clinically,protein markers are generally quantified by immunoassay.Compared with the mass concentration or molar concentration of protein,the immunoactivity concentration becomes the key to ensure test results accurate and comparable,and establishing an accurate absolute quantitative method of immunoactivity concentration of protein from the source has become the core of protein immunoactivity concentration.In recent years,Calibration free concentration analysis based on molecular interaction can meet the purpose of direct and accurate quantification of protein immunoactivity concentration.The method is based on the interaction of specific monoclonal antibodies and proteins without the need for external.It can accurately determine the concentration of antigen that binds to the antibody.In this paper,we studied the accurate quantitative method of immunoactivity concentration of myoglobin and transferrin,which is of great significance to ensure accurate and comparable detection results of myocardial diagnostic markers and guide the early diagnosis and subsequent treatment of myocardial injury.Firstly,the quantitative method of immunoactivity concentration of purified human myoglobin was studied.Three monoclonal antibodies were selected to quantify human myoglobin by CFCA method,the antibody immobilization conditions,level of immobilization,regeneration buffer and injection concentration were optimized.The methodological parameters were investigated and compared with the results of isotope dilution mass spectrometry.The immunoactivity concentrations obtained by three antibodies under optimized conditions were 2.985,2.912,and 3.032 mg/mL,which was in agreement with the concentration of human myoglobin obtained by isotope dilution mass spectrometry(IDMS)of 2.851 mg/mL.It changed the recognition that the concentrations of immunoactivity should be lower than the results of chemical analysis,indicated that by scouting antibodies with appropriate epitopes,the influence of advanced protein structure and modification on the immunoassay can be avoided,which can maintain the consistency of the result between immunoassay and isotope dilution mass spectrometry.It is helpful for developing certified reference materials with accurate value and interchangeability.The within-day repeatability of the method for quantifying immunoactivity concentration ranged from 2.50%to 4.23%,and the between-day reproducibility was 5.85%,indicating that the method had good repeatability and reproducibility.The binding parameters between antibody and protein were accurately measured with obtained immunoactivity concentration,the dissociation equilibrium constants were 8.97 × 10-9 M,1.4 × 10-10 M,and 1.55× 10-10 M,respectively.The research established a process for accurately measurement and calculation of immunoactivity concentration,and reveals the reasons for difference binding responses between antibodies and protein.It also indicates the relationship between the immunoactivity concentration,and the mass concentration or molar concentration based on the primary amino acid sequence,which is conducive to determine the interchangeability of standard materials on different immunoassay systems,and develop protein standard materials with accurate value and extensive use.Secondly,the method for measuring immunoactivity concentration of myocardial markers in complex matrices was studied.Two monoclonal antibodies were used to quantify transferrin in serum by CFCA method and compare with the results of purified transferrin.The immunoactivity concentrations of human serum transferrin in serum obtained by antibody MM03 and MM06 were 1.223 mg/mL and 1.064 mg/mL;the within-day precisions of the method were 6.44%～7.66%,6.57%～9.07%,respectively;The between-day reproducibilities were 7.07%and 8.13%.By comparison,the results obtained by antibody MM03 had better repeatability and reproducibility.The concentrations of purifed transferrin obtained by antibody MM03 was 1.282 mg/mL,the within-day repeatability ranged from 4.15%to 6.86%,and the between-day reproducibility was 6.12%.Compared with the concentration obtained by IDMS indicated that 53.84%of protein was active.Through these data,the immunoactivity concentration of transferrin in serum was converted to mass concentration with the result of 2.271 mg/mL,and the error was within 10%compared with the results of 2.464 mg/mL determined by biochemistry analyze,which explored a way to ensure the consistency of immunoassay results of antibodies with different affinity by conversion of immunoactivity concentration and the mass concentration measured by IDMS.The research established an accurate quantitative method for immunoactivity concentration of protein in complex matrices.It is anticipated the CFCA approach will be a quick and accurate quantitative method for myocardial injury markers in serum with features of label-free and high-throughput,and of great significance for the rapid diagnosis of cardiovascular diseases such as AMI.