| BackgroundCorona Virus Disease 2019(COVID-19)epidemic spread rapidly’,and the confirmed cases worldwide have exceeded 100 million.The epidemic is still spreading now.The disease progresses rapidly in the critical ill and,in severe cases,can be life-threatening.In response to the epidemic and secondary epide’mic of COVID-19 caused by Severe acute respiratory syndrome coronavirus 2(SARS-CoV2),a number of programs have been carried out worldwide,including disease diagnosis,contact tracing and asymptomatic infection rate monitoring,etc.Nucleic acid testing is still recognized as one of the preferred diagnostic method,which carries out etiological tests.In the seventh edition of COVID-19 diagnosis and treatment document issued by National Health Commission of the People’s Republic of China,antibody detection is regarded as one of the diagnostic methods.Antibody detection could be used as an auxiliary diagnosis for COVID-19,as well as to meet the needs of epidemiological investigation.ObjectiveIn this study,the receptor binding domain of spike protein was used as the target,aiming at setting up a time-resolved fluorescence immunochromatographic assay for rapid detection of antibody against SARS-CoV-2,providing a kind of technical means for auxiliary diagnosis and epidemiological investigation of COVID-19.Methods1.Identified the protein materials needed for fluorescence immunochromatographic assay by SDS-Polyacrylamide Gels Electrophoresis and Western blotting experiment.The laboratory reference preparation needed was prepared too.2.Process majorization was conducted by screening optimal raw materials(time-resolved fluorescent nanobeads,glass fiber cotton(raw material of conjugated pad),Nitrocellulose membrane),by optimizing immunochromatography technology(microspheres activator dosage,activation buffer pH,and amount of protein,etc.),as well as the formula of microsphere protective solution.3.Performance evaluation:the fluorescence immunochromatographic assay established in this study was evaluated for Cut-off value,precision,specificity and stability,and compared with the Guangzhou Wondfo Novel Coronavirus(2019nCoV)antibody detection kit(colloidal gold method)in methodology.Results1.The size of the protein was 27 kDa which was consistent with theoretical value and purity was high by SDS-Polyacrylamide Gels Electrophoresis and Western blot results.And the titer of the laboratory reference preparation met the requirement.2.A fluorescence immunochromatographic assay for detecting the antibody against SARS-CoV-2 was preliminarily established.The optimum raw materials were selected,and when activating 50 μL microspheres(1%solid content),the optimum amount of 10 mg/mL EDC was 40 μL,the optimum amount of 10 mg/mL NHS was 200 μL,the preferred activation buffer pH was 6.5±0.5,the best amount of label protein was 40μg,and we finally determined the best formula of microsphere protective solution.3.Performance evaluation of fluorescent immunochromatography strips:on the basis of the above optimal process,the Cut-off value of the strips was 0.027,the coefficient variation of the strips was up to grade,for the intra-batch and inter-batch coefficient of variation less than 10%.There was no obvious cross reaction with Mycoplasma pneumoniae,influenza A virus,influenza B virus and Epstein-Barr virus,which showed good specificity.The test strips prepared can be stored stably for 20 days under 37℃.A total of 61 clinical specimens were detected by Wondfo and self-made kit and the sensitivity of both means could reach 70%.Conclusion1.In this study,a set of available reference preparation for the assay was successfully prepared.2.In this study,a new recipe for fluorescence immunochromatography technology was obtained.The test strips prepared can be stored stably for 20 days under 37℃.3.A time-resolved fluorescence immunochromatographic assay for the rapid detection of antibody against SARS-CoV-2 was established in this study. |
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