Establishment of A Method for Serum Soluble fms-like Tyrosine Kinase-1 Based on Magnetic Nanoparticle Chemiluminescence Immunoassay and Its Preliminary Performance Evaluation
Establishment of A Method for Serum Soluble fms-like Tyrosine Kinase-1 Based on Magnetic Nanoparticle Chemiluminescence Immunoassay and Its Preliminary Performance Evaluation
Objective To establish a method for soluble fms-like tyrosine kinase-1(sFlt-1)in human serum based on magnetic nanoparticle chemiluminescence immunoassay and preliminarily evaluate its performance.Methods According to the principle of sandwich plate theory,sFlt-1 in serum reacted with biotinylated anti-sFlt-1 monoclonal antibody and acridinium ester labeled anti-sFlt-1 monoclonal antibody to form an immune complex of biotinylated antibody-antigen-acridinium ester labeled antibody.Through the reaction between biotin and streptavidin,the immune complex was captured by the magnetic nanoparticles coated with streptavidin.The acridinium ester labeled on the immune complex caused chemiluminescence in alkaline H2O2.The chemiluminescence intensity of the immune complex was used to calculate the concentration of sFlt-1 in serum,optimize the concentrations of antibodies,and establish a method for sFlt-l based on magnetic nanoparticle CLIA.The sensitivity,precision,linear range,interference,accuracy and method comparison of the established method were evaluated.A total of 100 clinical serum specimens were collected from pregnant women in the Ningbo Yinzhou Hospital of Traditional Chinese Medicine from February to August 2022.The established CLIA was used for detection,and the results were compared with the results of Roche electrochemiluminescence assay.Results The optimal concentrations of biotinylated antibody and acridinium ester labeled antibody in the reagent were ultimately determined to be 2 μ g/ml and 100 ng/ml,respectively.The limit of blank of this method was 1.67 pg/ml,the coefficient of variation(CV)of precision within and between batches was lower than 5％,and the linear range was 8.9～81 206pg/ml.When the concentrations of serum hemoglobin,bilirubin,fat emulsion and biotin were lower than 500 mg/dl,20 mg/dl,1 450 FTU and 50 μ g/L,respectively,there would be no significant interference with the detected results.The recovery rate of the method was 98％.The correlation coefficient(r2)between the established method and Roche electrochemiluminescence assay was 0.995 3.The difference between the two group was significant(P=0.000 3).Conclusion The established method have good preliminary performance and can be further applied to the auxiliary screening of preeclampsia.
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