Establishment And Application Of Immunoassay Technology Platforms Based On Time-resolved Fluorescence Microparticles

As a kind of new immune tracers,Time-resolved fluorescence microparticles possess excellent performance,such as super sensitivity,high stability and so on.In this study,time-resolved fluorescence microparticles as tracers were applied to lateral flow immunoassay and magnetic microparticles-based time-resolved fluoroimmunoassay,and its comprehensive and accurate evaluation of clinical applications was conducted.The experiment is mainly divided into three parts:(1)Development of the lateral flow strips based on CM-EUs for detecting urine NGAL;(2)Development of the lateral flow strips based on CM-EUs for detecting serum Anti-HBc;(2)Development of the magnetic microparticles-assisted time-resolved fluoroimmunoassay based on Time-resolved fluorescence microspheres for detecting AFP/Free ?-HCG.Each part above had covered the establishment,optimization and evaluation of the immunoassay,including dose-response curve,sensitivity,accuracy,precision and specificity.We also compared homemade reagents with similar reagents to evaluate the feasibility for clinical application.The first part:Under the optimum condition,the result showed that the analytical sensitivity was 0.30 ng/mL for NGAL and the linear range was 10?6000 ng/mL;The intra-assay coefficients of variation were between 5.23%?8.19%and the inter-assay coefficients of variation were between 7.02%?9.24%,the reproducibility and accuracy of the proposed assay were acceptable;A certain concentration of a-Microglobulin,hepatocyte growth factor,a 1-acidgly coprotein,matrix metalloproteinase-2 as sample to measure the cross-reactivity and the result was shown no significant cross-reactivity;Triglycerides mixtures,bilirubin,hemoglobin were added in corresponding control samples and no interferences were detected;114 urine samples were measured by ELISA method and the proposed method to evaluate the correlation,The result shown a good correlation coefficient(r = 0.9846,(P<0.001)),confirmed that two methods was significantly correlated.The second part:Under the optimum condition,the result showed that the analytical sensitivity was 0.31 IU/mL for Anti-HBc and the linear range was 0.63?640 lU/mL;Recovery of reference material was between 92.95%?97.12%;The intra-assay coefficients of variation were between 5.63%?7.79%and the inter-assay coefficients of variation were between 6.93%?9.82%,the reproducibility and accuracy of the proposed assay were satisfactory;Positive samples related to hepatitis b disease were measured the cross-reactivity and the result was shown no significant cross-reactivity;Triglycerides mixtures,bilirubin,hemoglobin were added in corresponding control samples and no interferences were detected;231 serum samples were measured by our developed immunoassay and a commercially available CMIA kit from Abbott,and coincidence rate of clinical specificity was 95.90%and sensitivity was 99.08%.Paired chi-square test was conducted with P=0.219,meaning that there was no significant difference in two kinds of methods for qualitative core antibody detection;Besides,two methods for the qualitative detection of core antibody consistency have a good consistency with Kappa = 0.948(P<0.001).Then,108 serum samples,both positive in homemade regent and CMIA kit,were measured by TRFIA method and the proposed method to evaluate the correlation,with a correlation coefficient r = 0.9605,(P<0.001),confirmed that two methods was significantly correlated.The third part:Under the optimum condition,the result showed that the analytical sensitivity was 0.10 IU/mL for AFP and the linear range was 2?480 IU/mL,and the analytical sensitivity was 0.35ng/mL for Free ?-HCG and the linear range was 3?200 ng/mL;Recovery of reference material was between 93.8%?100.36%for AFP and 97.8%?106.47%for Free ?-HCG,respectively;The intra-assay and inter-assay coefficients of variation were both below 10%,the reproducibility and accuracy of the proposed assay were acceptable;A certain concentration of CEA,CA125,CA15-3,CA19-9,HCG,LH,FSH,TSH and HSA as sample to measure the cross-reactivity and the result was shown no significant cross-reactivity;Triglycerides mixtures,bilirubin,hemoglobin were added in corresponding control samples and no interferences were detected;134 serum samples were measured by TRFIA kits and the proposed method to evaluate the correlation,with correlation coefficient r=0.9869(P<0.001)and r= 0.9904(P<0.001),confirmed that two methods was significantly correlated.We successfully established two immune technology platforms based on time-resolved fluorescence microparticles.In consideration of their high sensitivity,wide linear range,and rapid detection,the clinical application prospect of two immune technology platforms will very broad.