IgG Detection By Using Time-resolved Fluoroimmunoassay

Using complexes of rare earth elements with long fluorescent life、broad Stokes shiftand sharp characteristic peak as marker，time-resolved fluorescence immunoassay（TRFIA）can exclude background fluorescence efficiently through delay of measure time, whichmakes it more sensitive than other immunoassays.This work aims to establish the AP-based Enzyme Linked ImmunosorbentAssay（ELISA）、 Fluorescence Enzyme Immunoassay（FEIA） and time-resolvedfluorescence immunoassay（TRFIA）,which involves the coupling of BHHCT and antibody,comparing dictation limits of the three methods and applying this method to antibodydetection.The main contents in this paper are as follows:（1） Coupling BBCAP（ligand） and goat anti-mouse IgG, detect its immunocompetence.（2） Synthesis conjugate of BHHCT-goat anti-mouse IgG（GAM）, study the key procedureof TRFIA-Eu³⁺antibody labeling technology, discuss the factors affecting immune reactionsuch as Eu³⁺concentration、congugate dilution power、Eu³⁺placing time、placing time afteravailable Eu³⁺and diluted congugate mixing、photobleaching、temperature、Wallacwashing buffer and so on and ultimately definate1*10-5M as theEu³⁺usingconcentrate,1:20as the congugate dilution power,60min as the right placing time afteravailable Eu³⁺and diluted congugate mixing, Wallac washing buffer is more stable,photobleaching has no influence on fluorecsence detection，avoiding placing congugate athigh temperatures.（3） Using mouse-IgG as antigen, goat anti-mouse IgG（GAM） as antibody, establishcompetitive system of ELISA、 FEIA and TRFIA, with the sensitivity of ELISA:IC（20）=2.477μg/mL,IC（50）=21.115μg/mL;FEIA:IC（20）=2.522μg/mL,IC（50）=30.723μg/mL;TRFIA: IC（20）=0.113μg/mL, IC（50）=1.131μg/mL.