A Homogeneous Time-resolved Fluorescence Immunoassay Method For The Measurement Of Procalcitonin

Two rare earth fluorescent chelating agents were successfully synthesized and their structures were confirmed by ¹H-NMR and MS.The fluorescence properties of these two rare earth fluorescent chelating agents show that the characteristic excitation and emission peaks of Europium ions are maintained in both of the rare earth fluorescent chelating agents.The maximum excitation wavelength of Eu³⁺?bpy.bpy.py?CO₂H?₂ is 312 nm,and the maximum emission wavelength is 598 nm and 615 nm.The maximum excitation wavelength of Eu³⁺?bpy.bpy.py[CONH?CH₂?NH₂]₂ is 311 nm,and the maximum emission wavelength is 597 nm and 616 nm.The fluorescence intensity of two rare earth chelating agents is linearly correlated with their concentration and the R² reached 0.999.The linear equations are:Eu³⁺?bpy.bpy.py?CO₂H?₂:y=310x-565.98;Eu³⁺?bpy.bpy.py[CONH?CH₂?NH₂]₂:y=310x-1930.The lifetime of fluorescence is 1064?s and 398?s,respectively,and the quantum yields are10.1%and 12.1%,respectively.Both of the rare earth chelating agents have good fluorescence characteristics and meet the needs of time-resolved fluorescence immunoassay.Modified on the basis of Eu³⁺?bpy.bpy.py[CONH?CH₂?NH₂]₂,Eu³⁺?bpy.bpy.py[CONH?CH₂?NHCOCH₂OCH₂COOH₂]₂ was synthesized and confirmed by mass spectrometry.The maximum excitation wavelength is 312 nm,and the maximum emission wavelength is 598 nm and 615 nm.The linear equation of fluorescence intensity and concentration is y=310x-1041.4,R²=0.999.DCC/NHS was used as dehydrating agent to combine chelating agent with antibody under the catalysis of DMAP.After optimizing the labelingconditions,thebestlabelingconditionsforEu³⁺?bpy.bpy.py[CONH?CH2?NHCOCH2OCH2COOH2]2 and antibody were found to be:molar ratio of chelating agent to DCC/NHS is 1:4:6;reaction for 6h under room temperature;reaction with antibody protein at molar ratio of 20:1 for 10 hours,rare earth fluorescent chelator labeled antibody with labelled ratio of 10 can be obtained.The titer of antibody protein was 1:800 after confirmation by ELISA.Construction of a time-resolved immunofluorescence assay for procalcitonin detection using labeled anti-PCT detection antibody and anti-PCT coating antibody.The experimental results show that The optimum dilution ratio of anti-PCT coating antibody is 1:200;the optimum dilution ratio of anti-PCT detection antibody is 1:400;the optimum incubation time was 60 minutes;in the concentration range of 0.5-50ng/mL,the intensity of fluorescence signal is linearly correlated with the concentration of antigen and the linear equation is y=2552.6x+4.8222,R²=0.9928;the sensitivity is 0.5ng/mL;CV<10%;and there is no cross reaction with common clinical markers such as AFP,CEA and CRP.