Study On Immunolabeling Technology Of Rare Earth Cryptate

ObjectiveThe aim of this study was to synthesize an ideal rare earth cryptate and use it for labeling of monoclonal antibody of alpha-fetoprotein(AFP-McAb),exploring optimal conditions of labeling,which was to test the effect of self-synthesized rare earth cryptate used for labeling of antibodies.This study would provide a reference for labeling of biomolecules and lay a foundation for the further construction of homogeneous time-resolved fluorescence immunoassay.MethodDiethyl 2,6-dimethylpyridine-3,5-dicarboxylate was used as raw material to optimally synthesize Li+(?)bpy.bpy.py(CO2Et)2.The reaction system of acetonitrile or methanol was used to synthesize Eu3+(?)bpy.bpy.py(CO2Et)2 that was discussed about the difference of spectral properties.Eu3+(?)bpy.bpy.py(CO2Et)2 was hydrolyzed to form Eu3+(?)bpy,bpy,py(CO2H)2 that was used as a tracer.Eu3+(?)bpy.bpy.py(CO2H)2 was activated by EDC and NHS to label AFP-McAb.In order to obtain optimum activation time,coupling time,reaction pH and reaction ratio of europium cryptate,the effect of labeling was evaluated by fluorescent intensity,labeling rate and relative immune activity of labeled antibody.ResultBy changing the concentration of initial reactants,the yield of Li+(?)bpy.bpy.py(CO2Et)2 was obviously increased to 51%.The excitation spectra(maximum excitation wavelength of 310 nm),emission spectra(maximum emission wavelength of 616 nm)and quantum yield of Eu3+(?)bpy.bpy.py(CO2Et)2 obtained from two reaction systems were basically the same.The fluorescence intensity depended linearly on the concentration of Eu3+(?)bpy.bpy.py(CO2Et)2 in the range of 4.0×10-8-2.5×10-5 mol/l and the coefficient of correlation were 0.99373 and 0.98665,respectively.The fluorescence lifetime were 825?s and 830?s,respectively.The fluorescence intensity of Eu3+(?)bpy.bpy.py(CO2Et)2 synthesized by the reaction system of acetonitrile was slightly high.The optimal activation time,coupling time,reaction pH and reaction ratio of europium cryptate-labeled AFP-McAb were 24h,24h,9,60:1,respectively.ConclusionBoth reaction systems can synthesize bifunctional europium cryptate with high fluorescence intensity and strong stability,which can be used for the labeling of AFP-McAb.The labeled antibodies have strong fluorescence intensity and good immunoreactivity,which lay a foundation for the establishment of homogeneous time-resolved fluorescence immunoassay.