Establishment Of Chemiluminescence Immunoassay Method For Plasmin-α2-plasmin Inhibitor Complex

Plasmin-α2-plasmin inhibitor complex（Plasmin-α2-plasmin inhibitor complex,PIC）is a marker of thrombosis,which can provide evidence for the diagnosis,prescribing medicine and prediction of thrombosis.Currently,enzyme-linked immunosorbent assay（ELISA）and chemiluminescence immunoassay（CLIA）are commonly used to detect PIC.CLIA method has higher sensitivity and stability than ELISA.However,the domestic PIC chemiluminescence detection market is monopolized by the Japanese company Sysmex.Therefore,this research aims to establish the research and development of chemiluminescence detection PIC,which can achieve import substitution and break the monopoly,ultimately reduce the medical expenses of the general public.In this study,the preparations of the PIC antigen and its antibody were studied.At the same time,the CLIA method was established to detect PIC,and the reaction system was optimized.Then the performance of the detection method was evaluated,and the reference interval and clinical performance of the detection method were finally analyzed.The main findings are as follows:（1）Preparation of PIC and its precursor molecule Plasminogen（Plg）It was determined that the optimal activation condition for preparing PIC was 2.0 mg urokinase for 100 mL plasma with 30 min at temperature of 37℃.After affinity chromatography,2.5 mg PIC was finally obtained for 100 mL plasma.Plg was used for the screening of PIC monoclonal antibodies,and 6.5 mg Plg was collected by affinity chromatography.The purity of PIC and Plg were both above 90%through SDS-PAGE.Using PIC as the antigen,7 monoclonal antibodies were obtained.（2）The optimization of reaction system7μg·mL^-1 biotin-labeled bovine serum albumin（BSA）and 13μg·mL^-1 were used to prepare streptavidin（SA）coated plates.The dilution matrix of PIC was determined to be 0.02g·L^-1 sucrose,0.03 g·L^-1 BSA,0.2 mol·L^-1 PB pH 7.5.Seven monoclonal antibodies were screened by antibody pairing experiments.The results showed that the best pairing combination was obtained when PB₃ was used as the coated capture antibody and PM₂ was used as the enzyme-labeled detection antibody.The square matrix test showed that the system had the best effect when the antibody was 30 mL 0.8μg·mL^-1 PB₃-Biotin and 30 mL 0.3μg·mL^-1 PM₂-HRP.The working system were reaction time of 15 minutes,washing the plate 4 times,using 0.3%luminol（LMO）and 0.05%borax as substrate A and substrate B,and testing within 3 minutes after the substrate was added.（3）Performance evaluationThe detection limit was 0.08μg·mL^-1,and the linear range was 0.06～42.00μg·mL^-1.In terms of repeatability,the intra-batch and inter-batch CV were both below 10%.When less than5.0 g·L^-1 bilirubin and hemoglobin and less than 0.5 g·L^-1 triglycerides were added to the serum,there was no interference with the determination method.It was confirmed that the PIC detection method can maintain stable performance for 11 days at 37℃,and the shelf life was17 months at 4℃.When the sample concentrations were between 0 and 78.17μg·mL^-1,the hook effect did not appear in this detection method.（4）Method traceability researchThe traceability with Sysmex’s PIC detection method was completed.The total uncertainty of the process was low at 8.96%,and the traceability process had high credibility.（5）Reference interval and clinical evaluationThe reference value of PIC range was 0 to 0.8μg·mL^-1.264 clinical serum samples were simultaneously tested by CLIA method and Sysmex’s PIC detection method,and the results showed that the correlation coefficient of the two determination methods was 0.9806,which had good consistency and high coincidence rate.Therefore,there was no statistical difference between the two determination methods（P>0.05）.This study established the CLIA method to determine PIC.The results showed that the performance of this method and clinical evaluation met the research requirements,so this method can be used to detect PIC content.