Development And Application Of Novel Highly-Sensitive Immuno-based Point-of-Care Technologies

In Vitro Diagnosis(IVD)plays an important role in disease prevention,disease diagnosis,treatment monitoring,prognostic observation,drug screening and healthy state evaluation.About two-thirds of medical decisions are made based on IVD information.The development of IVD techniques is the key element to improve decision making on disease prevention,diagnosis,treatment.The high sensitivity and fully automation of IVD have been achieved in central laboratory.With the drawbacks of the need of expensive equipment,long test turnaround time,highly-trained personnel,these techniques could not meet the need of infectious diseases on-site detection and critical illness rapid diagnosis.These techniques are not suited for use at home,either in primary health care units.Point-of-care Testing(POCT)products lower the requirements of the hardware,they are suitable for infectious diseases rapid on-site detection.They could be used for community health centers,clinics and other primary health care units,and self-testing at home.In clinical practice,some biomarkers(especially infectious diseases test markers)often require high analytical sensitivity but without quantification.The traditional rapid immunodiagnostic reagent are mainly based on colloidal gold technology.The colloidal gold based products lack detecting sensitivity,and therefore there is an urgent need to establish a highly sensitive immunoassay method for rapid on-site detection.On the other hand,more and more immune targets require rapid quantitative on-site detection.The colloidal gold based products are difficult to use in these cases because they lack sensitivity,enough quantitative range and quantitative accuracy.This dissertation focuses on developing lateral flow immunoassay-based methods for rapid,sensitive qualitative detection and rapid,sensitive quantitative detection.In the first part of this study,a novel platform was developed based on HRP labelling by designing a specific layout,specific cartridge for strip,and the substrate system and the blocking reagents were studied.No washing steps,color development,terminating step were required,reaction would be finished within 30min.And after color film background clean,clear bands,not because of the film and the negative control background false positive results to facilitate interpretation of the results.Due to the HRP atalytic cascade effect,40 times higher sensitive than colloidal gold was realized by HRP-labeled strip,along with good specificity.Flu A/B antigen detecting strip and RSV antigen detecting strip were developed on the platform.For the detection of influenza strains,the analytical sensitivity of Flu A strip reach ed 0.00025 hemagglutination(HA),for influenza B strain was 0.01 HA.The evaluation was done by a total of 1487 clinical swab specimens using PCR methods as reference.The sensitivity is 77.52%(69.58%-83.87%)and the specificity is 99.83%(99.37%-99.95%)for flu A for all group,as the sensitivity is 90.16%(80.15%-95.41%)and the specificity is 99.65%(98.73%-99.90%)for nasopharyngeal swabs,and the sensitivity is 66.18%(54.34%-76.29%)and the specificity is 100%(99.34%-100.00%)for nasal swabs.For influenza B,the total sensitivity is 71.17%(63.79%-77.57%).When using the nasopharyngeal swab specimens,the detection sensitivity is 82.56%(73.20%-89.14%)and the specificity is 100%(98.70%-100.00%).The sensitivity is 58.44%(47.29%-68.79%)and the specificity is 99.68%(98.84%-99.91%)using the nasal swabs.RSV strip was evaluated using 1078 clinical samples.The agreements between strip and reference is 96.03%(94.34%?97.32%)with nasopharyngeal aspirate fluid as specimen,90.52%(86.94%?93.38%)with nasopharyngeal swabs as specimen.The total agreement is 94.25%(92.69%?95.56%)?The second part was aimd to establish a time-resolved fluorescence-labeled microspheres based highly sensitive immunochromatographic quantitative detection platform.Covalent conjugation was realized by EDC activating method.We developed a line scanning fluorescence reader,its optical module was using optical fiber materials.The area ratio between test line and control line was chosen to be original quantitative value.The detection CV was under 3%for the reader.CRP and PCT quantitative strips were developed.CRP can be quantified in the range of 1mg/L-100mg/L.PCT quantitative range is between 100pg/mL-lOng/mL,both of which cover the range of clinically significant.Correlation analysis were also studied,the correlation coefficient R2 is 0.9934 and 0.9204 respectively.TB-IGRA quantitative strips can be quantified in the range of 12.5pg/mL-400pg/mL,it's consistent with the commercial ELISA kits.29 specimens were used for correlation analysis,R2 is 0.9373.The clinical evaluation result using 1113 samples reached a agreement rate of 93.87%(92.30%-95.14%)compared to reference reagent.Based on double antigen sandwich method,HCV qualitative strip was developed,3-5 times more sensitive was observed by comparison with commercial ELISA.42 serums positive confirmed by CHIRON RIBA were from clinical laboratory center.The sensitivity of HCV antibody strip was 47.62%(33.36%-62.28%),21.43%(11.71%-35.94%)for Roche diagnostics and Beijing Wantai sandwich and indirect ELISA reagent,was 19.05%(9.98%-33.30%)and 19.05%(9.98%-33.30%).In summary,the present study has developed a high sensitivity,qualitative immunoassay platforms and a highly sensitive a quantitative detection platform,based on the platform we've developed some rapid diagnostic reagents for a variety of clinical urgent needs,with broad application in clinical practice prospect.