药物分析期末.ppt

Time-resolved fluorescence immunoassay (TRFIA) Main content The definition of TRFIA The principle of TRFIA The advantages of TRFIA The clinical application of detecting HBV infection Definition TRFIA is a analysis that use trivalent rare earth ions and chelating agents as a tracer, instead of fluorescent substances, isotopes or enzyme, marking up protein, hormone, antibody, nucleic acid probes or biological activity cells. When the reaction occurs, TRF instrument detect the fluorescent intensity of final product. According to the fluorescence intensity to determine concentration of the substances. Unique fluorescent marker of rare earth metals- Lanthanide Total 15 kinds of lanthanide elements(trivalent element), 4 elements used in TRFIA 铕(Eu)、钐Sm)、镝(Dy)、铽(Te) Eu3+ is most widely application Sm3+ as second option can be used for double labeling or labeling Different trivalent rare earth ions have different emission spectra and fluorescence lifetime. It can be used to double labeling and multi-tag and detect two or more antigens or antibodies simultaneously. Characteristics of lanthanide fluorescence : Stokes shift is large Eu: emission 613nm, excitation 340nm Fluorescence near 280nm Fluorescence lifetime is long Lanthanide chelates (60 ~ 900 us) Eu: 714us Fluorophores of common immunofluorescence : 1 ~ 100nsProtein fluorescence: 1 ~ 10ns, easy to quenching Fluorescence specificity Emission band is narrow, even less than 10nm Dissociation - Enhancement technology enhance intensity 100 million times Delaying measurement time Fluorescence of short-lived sample decay completely Detection of rare earth ion chelate fluorescence signal Eliminated non-specific fluorescence from the sample, reagents This is the“Time-resolved” Dissociation enhanced lauthanide fluoroimmunoassay DELFIA is unique After immune response completed, part of the marker binding to the solid phase carrier.