Establishment Of Fluorescent Immunochromatography Assay For Detection Of SARS-CoV-2 Antibody

BackgroundSo far,corona virus disease 2019(COVID-19)has infected more than 1.5 million worldwide and caused nearly 3.3 million deaths.The disease spreads rapidly and still has no specific treatment in recent days.There are effective ways to prevent further spread of the virus by early diagnosis and timely management.At present,there are three main diagnostic methods of COVID-19: viral gene detection,antibody detection and antigen detection.Related methods develop rapidly,but these still caused a series of problems likes high cost,wasting time and low sensitivity.Based on the situation,it is urgent to establish a detection method to make up for the shortcomings.ObjectiveTo establish a detection method by time resolved fluoroimmunoassay for evaluating SARS-CoV-2 antibody level in serum,based on severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)recombinant N protein.This method will provide a new method for the primary screening and auxiliary diagnosis of COVID-19.Methods1.Identification of recombinant antigen: Using sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE)and Western Blot(WB)to detect the expression of the target protein base on SARS-CoV-2 recombinant N protein(NC).2.Polyclonal antibody preparation: The New Zealand white rabbit was immunized with recombinant protein NC,and the obtained antiserum was purified.The purified polyclonal antibody was identified by Western blot,and its titer was determined.Meanwhile,using this antibody to prepare indoor reference products.3.Establishment of fluorescence immunochromatographic assay for SARS-CoV-2antibody: Screening two kinds of raw materials of glass fiber membrane and nitrocellulose membrane.Optimizing the coupling time,amount of activator,p H of buffer,amount of labeled protein and other aspects in the preparation process.Preparing the test strips by using the optimized conditions,and evaluating its performance in terms of cut-off value,precision,specificity and accelerated stability.Results1.By SDS-PAGE,the position of the obtained target band was consistent with the molecular weight of NC antigen.By Western Blot,the protein was able to specifically bind to the SARS-CoV-2 N protein(NP)antibody.2.The titer of purified rabbit-derived polyclonal antibody was improved fourfold compared to before purification,by enzyme linked immunosorbent assay(ELISA).And also it could correctly recognize recombinant antigen NC and native antigen NP as identified by WB,and a series of indoor reference products were successfully prepared.3.Through raw material screening and process optimization,a rapid method for evaluating SARS-CoV-2 antibody levels in serum was finally established.In this method,the cut-off of T/C value was 0.05,and the cut-off of T value was 1008.The intra-assay precision and the inter-assay precision met the requirements,and there was no cross reaction with mycoplasma pneumoniae,Epstein-Barr virus,influenza A virus and influenza B virus.Compared with the marketed kit,the coincidence rate could reach 97.04%.Conclusions1.The specificity of the recombinant antigen is determined after testing.And the antigen is highly pure,up to more than 99%.2.The polyclonal antibody with high titer and good specificity is obtained.A set of indoor reference products for antibody detection is established.3.The fluorescence immunochromatographic assay for the detection of SARS-CoV-2antibody is preliminarily established.