Sensitive ELISA To Detect SARS-CoV-2 Spike Antigen Based On Specific Peptide Screened From Phage Display And Tyramine Signal Amplification

The rapid spread of the severe acute respiratory syndrome coronavirus 2（SARS-CoV-2）has caused catastrophic public health problems and economic crises worldwide.Spike protein（SP）is the most important membrane protein on the surface of this virus and a key protein for viral invasion into cells;Therefore,it is one of the most valuable antigenic markers for the diagnosis of SARS-CoV-2.In this study,we used the Ph.D.-12 phage random peptide library for biological screening of SARS-CoV-2 SP antigen,and obtained six different peptide sequences,which were verified for binding ability and specificity,and finally confirmed that one of the phages expressed WNLDLSQWLPPM peptide sequences had high affinity and good specificity.The binding site of the peptide was determined by molecular docking to be located in the S2subunit of SARS-CoV-2 SP.A direct mode p-ELISA assay was constructed to detect SARS-CoV-2 antigen based on peptide as the capture probe.To improve the sensitivity of the assay,the main experimental parameters of the p-ELISA were optimized,including a series of experimental parameters such as coating buffer,coating concentration,antigen capture time and antibody dilution.Based on the optimized optimal experimental conditions,SARS-CoV-2 SP antigen as low as 40 pg/m L（0.28 p M）can be detected in direct mode.Using this method for pseudovirus detection,pseudoviruses down to 60 TCID₅₀/m L can be detected in nasal swab samples.A signal amplification assay system was successfully constructed to further improve the sensitivity of the assay.The tyramine signal amplification technique was applied to the direct detection mode p-ELISA,resulting in a significant enhancement of the detection signal.In the SARS-CoV-2 SP antigen assay,the primary signal amplification achieved a limit of detection of 2 pg/m L,which was 20 times more sensitive than the direct detection mode.The secondary signal amplification achieved a limit of detection of0.4 pg/m L,which was 100 times more sensitive.The proposed p-ELISA assay in signal amplification mode could detect pseudoviruses in healthy nasal swab samples as low as 3TCID₅₀/m L in the secondary signal amplification mode.These results demonstrate that the method can effectively detect virus content in complex systems and is a reliable technique for detecting SARS-CoV-2 antigen.Moreover,the signal amplification technique suggested in this investigation is versatile and can be readily implemented in other ELISA formats with exceptional adaptability.