Preliminary Study And Application Of Time-resolved Fluorescence Immunochromatography

Research Background:With the rapid development of in vitro diagnostic technology,immunochromatography（ICA）has become one of the important methods applied to rapid bedside detection（POCT）.At present,the most representative application of immunochromatography technology is colloidal gold immunochromatography test paper,which can be used for rapid qualitative detection of specific antigens.However,due to the influence of tracer,colloidal gold immunochromatography can not achieve quantitative detection.Time-resolved fluorescence immunoassay（TRFIA）is a quantitative detection and analysis method that uses rare earth element ions as antibody tracer and can distinguish specific fluorescence from non-specific fluorescence through time-delay detection.Based on this technology,the time-resolved fluorescence immunochromatography technology（TRFICA）is a combination of time-resolved fluorescence immune principle and immunochromatography technology.marker prepared by lanthanide elements is used to replace colloidal gold and other substances to label biological macromolecules such as antibodies.After specific reaction occurs in the system constructed with solid phase chromatography material,The detection area is scanned by a time-resolved instrument.The lanthanides of the detection line（T-line）and the quality control line（C-line）are excited and emit fluorescence.The fluorescence intensity is measured and converted into a digital signal,so as to realize the purpose of rapid quantitative analysis.markers are an important part of time-resolved fluorescence immunochromatography technology.By markers,Eu³⁺and biomacromolecules can be linked together to form the chelating compound of"biomacromolecule-CA-Eu³⁺",which can be used for the labeling of targets in time-resolved fluorescence immunoassay.There are many kinds of markers and their physical and chemical properties differ greatly.Therefore,it is very important to select appropriate markers for the suggestion of time-resolved fluorescence immunochromatography.Research methods:This study mainly includes two parts.The first part is to screen the five kinds of markers prepared in the laboratory in the previous stage,and mainly investigate the solubility,fluorescence intensity,protein labeling ability,optimal incubation time and temperature,and cross-linking molar ratio of each marker,so as to screen out a marker with high fluorescence intensity and high labeling efficiency.The second part is to optimize the processing conditions of sample pads and binding pads by selecting NC film,quality control line and coating concentration of test strips based on the principle of time-resolved fluorescence immunoassay technology,using the screened markers and hepatitis B monoclonal antibody as the target.A time-resolved immunochromatographic method（time-resolved fluorescence immunochromatographic paper）for hepatitis B detection was established,and its performance was analyzed and measured.Research results:The results showed that the best reaction solution of marker A was PBS（0.01 M p H 7.5）,the best reaction solution of marker B was PBS（0.01 M p H 8.5）,and the best reaction solution of marker C was PBS（0.01 M p H 8.5）.The best reaction solution of BHHCT was CBS（0.05M p H 8.9）,and the best reaction solution of BHHST was CBS（0.1M p H 8.9）.In the best reaction solution,the labeling and luminescence efficiency of the marker BHHCT was the highest,and the best cross-linking molar ratio of marker A,marker B,marker C and BHHST was 1:30.The optimum cross-linking molar ratio of BHHCT was 1:60,and the optimum incubation time and temperature of protein were 2 h and 37℃.Sephadex G-25 was selected as the chromatographic gel,and Tris-HCl（0.05M p H8.0）as the eluent.Based on the above results,BHHCT was selected as the best marker.BHHCT was used as marker to establish hepatitis B time-resolved fluorescence immunochromatography.The sample pad was soaked in 0.01M PBS（containing 1%BSA and 5%Tween20）,and the sample pad was soaked in the stock solution after the chromatography,CN140 was selected as the NC membrane,and the detection line was coated with hepatitis B polyclonal antibody at the concentration of 1.5 mg/m L.The quality control line was coated with 1 mg/m L sheep anti-mouse antibody,and the optimal detection time was 25 min.The fitting curve y=2687.8x-10818,R²=0.9941 between the ratio of antigen concentration T/C and T-line fluorescence intensity showed good linearity,low sensitivity,good specificity,no cross reaction,and good repeatability.The strip has good stability.Conclusions:In this study,a rare earth marker BHHCT with the best luminescence ability was selected from the five markers,and a time-resolved fluorescence immunochromatography method（antigen detection strip）was established for hepatitis B detection by using this marker.The strip has certain specificity and stability,and can be used for quantitative analysis of hepatitis B antigen effectively.This study will provide a new idea for the research and product development of time-resolved fluorescence immunochromatography.