Journal: Nature Communications

Article Title: A CRISPR-based ultrasensitive assay detects attomolar concentrations of SARS-CoV-2 antibodies in clinical samples

doi: 10.1038/s41467-022-32371-4

Figure Lengend Snippet: a Schematic illustration of the workflow of the UCAD assay. b Sequence design of the dsDNA template for triggering the collateral cleavage activity of CRISPR-Cas12a, as well as the strategy to split the dsDNA template into TS and NTS probes for UCAD. c Proximity binding of the two UCAD probes to the same anti-RBD antibody through affinity motifs enhances the T m of the duplex domain from 10 to 46 °C and thus enables the stable binding between TS and NTS probes. d , e Kinetic curves for the detection of anti-RBD IgG ( d ) and IgM ( e ) in commercially purchased certified anti-SARS-CoV-2 IgG/IgM positive and negative serum samples using UCAD. f A calibration curve for the detection of anti-SARS-CoV-2 spike protein RBD human mAb (clone: OTIH401) in the range of 1 aM to 1 pM was established by plotting fluorescence intensity at 40 min of the CRISPR-Cas12a reaction as a function of target concentrations, which was also compared with UCAD without RPA amplification. Each concentration has replicated measurements ( n = 2). g , h Detection of anti-RBD IgG ( g ) and IgM ( h ) in certified anti-SARS-CoV-2 IgG/IgM positive serum diluted in negative human serum with dilution factors from 1 to 100,000 using both UCAD and commercial ELISA kits. Each concentration has replicated measurements ( n = 2). Source data are available in the Source Data file.

Article Snippet: SARS-CoV-2 Spike RBD human monoclonal antibody (Cat #: TA190325, clone name: OTIH401) and SARS-CoV-2 N protein human monoclonal antibody (Cat #: TA190323, clone name: OTIH1G5) were purchased from Origene, Beijing, China.

Techniques: Sequencing, Activity Assay, CRISPR, Binding Assay, Fluorescence, Amplification, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Nature Communications

Article Title: A CRISPR-based ultrasensitive assay detects attomolar concentrations of SARS-CoV-2 antibodies in clinical samples

doi: 10.1038/s41467-022-32371-4

Figure Lengend Snippet: a Detection of the target anti-human RBD and closely related nontargeted anti-MERS-CoV spike protein RBD human mAb (clone: m336), anti-SARS-CoV-2 nucleocapsid (N) protein human mAb, and anti-SARS-CoV-2 spike protein RBD rabbit pAb at a concentration of 10 fM using UCAD. Each sample was measured twice in two independent experiments. b , c Evaluation of the specificity of UCAD for anti-RBD IgG ( b ) and IgM ( c ) against anti-SARS-CoV in clinical sera from three SARS patients in 2003. Each serum sample was measured twice in two independent experiments. The UCAD signals of anti-SARS-CoV positive sera were significantly lower than the anti-SARS-CoV-2 positive serum ( p = 0.0004 for IgG, p = 0.0025 for IgM). Ordinary one-way ANOVA were used to compare the difference between multiple groups. d Schematic illustration of the modularity of UCAD for varying mutants of RBD and the N protein of SARS-CoV-2 by switching the recognition motif on the TS probe. e Heatmap of the detection of 10 fM wild-type (WT), Delta specific, and Omicron specific anti-RBD human mAb by using WT RBD, Delta RBD, and Omicron RBD (B.1.1.529) modified TS probes. f Heatmap of the detection of 10 fM WT RBD human mAb and anti-N protein human mAb with WT RBD and N protein modified TS probes. Source data are available in the Source Data file. ns: p > 0.05, *** p ≤ 0.001.

Article Snippet: SARS-CoV-2 Spike RBD human monoclonal antibody (Cat #: TA190325, clone name: OTIH401) and SARS-CoV-2 N protein human monoclonal antibody (Cat #: TA190323, clone name: OTIH1G5) were purchased from Origene, Beijing, China.

Techniques: Concentration Assay, Modification

Journal: Nature Communications

Article Title: A CRISPR-based ultrasensitive assay detects attomolar concentrations of SARS-CoV-2 antibodies in clinical samples

doi: 10.1038/s41467-022-32371-4

Figure Lengend Snippet: a Schematic illustration of lateral flow readout for UCAD for the detection of anti-SARS-CoV-2 IgG and IgM. b The design of the lateral flow strip that captured all anti-FAM labeled AuNPs at C-line in the absence of the target antibody. In the presence of ant-SARS-CoV-2 IgG or IgM, the FAM-Dig dual-labeled reporters were cleaved by Cas12a-crRNA so that AuNPs could escape the capture at C-line and accumulated at T-line through the immobilized secondary antibody. c Visual detection of 15 positive clinical sera and 15 pre-pandemic sera by UCAD integrated with lateral flow strips.

Article Snippet: SARS-CoV-2 Spike RBD human monoclonal antibody (Cat #: TA190325, clone name: OTIH401) and SARS-CoV-2 N protein human monoclonal antibody (Cat #: TA190323, clone name: OTIH1G5) were purchased from Origene, Beijing, China.

Techniques: Stripping Membranes, Labeling

Journal: Analytical Chemistry

Article Title: Quantitation of Trastuzumab and an Antibody to SARS-CoV-2 in Minutes Using Affinity Membranes in 96-Well Plates

doi: 10.1021/acs.analchem.1c03654

Figure Lengend Snippet: Calibration curve for the analysis of an antispike mAb added to 5% pooled patient sera. Assays comprise binding of antispike mAbs to an RBD-his-modified membrane in a 96-well plate, rinsing, binding of Cy5-labeled secondary antibody, rinsing, and measurement of fluorescence at 670 nm. Error bars represent the standard deviation from three replicate experiments. The dashed curve shows a four-parameter logistic fit to the data. Data were obtained with 24 different wells, three different wells for each concentration, and analyses on multiple days.

Article Snippet: Anti-SARS-CoV-2 spike receptor-binding domain (RBD) antibody [Chimeric mAb, Human IgG1 (S1N-M130)] was purchased from AcroBiosystem, and SARS-CoV-2 Spike RBD protein (his-tagged at the C-terminus, CHO-expressed) was purchased from GenScript.

Techniques: Binding Assay, Modification, Labeling, Fluorescence, Standard Deviation, Concentration Assay

Journal: bioRxiv

Article Title: Robust neutralization assay based on SARS-CoV-2 S-bearing vesicular stomatitis virus (VSV) pseudovirus and ACE2-overexpressed BHK21 cells

doi: 10.1101/2020.04.08.026948

Figure Lengend Snippet: Generation of VSV pseudotypes bearing SARS-CoV-2 spike proteins. (A) The difference between SARS-CoV-2-S and SARS-CoV-2-Sde18. (B) The procedure of producing VSV pseudotypes bearing SARS-CoV-2 spike proteins.

Article Snippet: SARS-CoV-2 spike specific antibody was generated by immunized Balb/c mouse with SARS-CoV-2 spike protein (RBD, Fc Tag, 40592-V05H, Sino Biological inc).

Techniques:

Journal: bioRxiv

Article Title: Robust neutralization assay based on SARS-CoV-2 S-bearing vesicular stomatitis virus (VSV) pseudovirus and ACE2-overexpressed BHK21 cells

doi: 10.1101/2020.04.08.026948

Figure Lengend Snippet: Comparison of the infectious efficiency of pseudotypes in various cell lines. VSVdG virus bearing spike protein of SARS-CoV-2 or G protein of VSV were harvested and the infectivity of these recombinant virus were tested in different cell lines, including Vero-E6, BHK21, 293T and BHK21-hACE2. The fluorescence was detected (A) and GFP positive cell number (B) was counted using Opera Phenix 12 h post infection.

Article Snippet: SARS-CoV-2 spike specific antibody was generated by immunized Balb/c mouse with SARS-CoV-2 spike protein (RBD, Fc Tag, 40592-V05H, Sino Biological inc).

Techniques: Infection, Recombinant, Fluorescence

Journal: bioRxiv

Article Title: Robust neutralization assay based on SARS-CoV-2 S-bearing vesicular stomatitis virus (VSV) pseudovirus and ACE2-overexpressed BHK21 cells

doi: 10.1101/2020.04.08.026948

Figure Lengend Snippet: Comparison of the packaging efficiency of VSVdG-SARS-CoV-2-Sdel18 in various cell lines. Vero-E6, BHK21 and 293T cells were used to package VSVdG-SARS-CoV-2-Sdel18 virus. The left picture shows the cell used to package recombinant virus, recorded 48h post infection of VSVdG-EGFP-G. The right figures show the infectivity of virus produced by three cell lines. The harvested virus was diluted and tested in BHK21-hACE2 cell. The fluorescence was detected (A) and GFP positive cell number (B) was counted using Opera Phenix 12 h post infection.

Article Snippet: SARS-CoV-2 spike specific antibody was generated by immunized Balb/c mouse with SARS-CoV-2 spike protein (RBD, Fc Tag, 40592-V05H, Sino Biological inc).

Techniques: Recombinant, Infection, Produced, Fluorescence

Journal: bioRxiv

Article Title: Robust neutralization assay based on SARS-CoV-2 S-bearing vesicular stomatitis virus (VSV) pseudovirus and ACE2-overexpressed BHK21 cells

doi: 10.1101/2020.04.08.026948

Figure Lengend Snippet: Time course of EGFP expression after VSVdG-SARS-CoV-2-Sdel18 infection. BHK21-hACE2 cell was infected with VSVdG-SARS-CoV-2-Sdel18 virus. The fluorescence was detected (A) and GFP positive cell number (B) was counted using Opera Phenix at different time point post infection.

Article Snippet: SARS-CoV-2 spike specific antibody was generated by immunized Balb/c mouse with SARS-CoV-2 spike protein (RBD, Fc Tag, 40592-V05H, Sino Biological inc).

Techniques: Expressing, Infection, Fluorescence

Journal: bioRxiv

Article Title: Robust neutralization assay based on SARS-CoV-2 S-bearing vesicular stomatitis virus (VSV) pseudovirus and ACE2-overexpressed BHK21 cells

doi: 10.1101/2020.04.08.026948

Figure Lengend Snippet: VSVdG-SARS-CoV-2-Sdel18 based system for screening of neutralizing mAbs. A Neutralizing antibodies were screened from 35 antibodies. The culture supernatant of 35 monoclonal hybridoma cells were incubated with VSVdG-SARS-CoV-2-Sdel18 virus and then added the mixture to BHK21-hACE2 cell. The fluorescence was detected using Opera Phenix 12 h post infection. The GFP positive cell number was also counted to calculate the inhibition rate (B). C The IC50 of 7 selected neutralizing antibodies for antiviral activity were also analyzed. The 7 selected neutralizing antibodies were purified and diluted to different concentration, then incubated with VSVdG-SARS-CoV-2-Sdel18 virus for an hour and added to BHK21-hACE2 cell. The GFP positive cell number was counted using Opera Phenix 12 h post infection to calculate the inhibit ratio. The IC50 was analysed by nonlinear regression.

Article Snippet: SARS-CoV-2 spike specific antibody was generated by immunized Balb/c mouse with SARS-CoV-2 spike protein (RBD, Fc Tag, 40592-V05H, Sino Biological inc).

Techniques: Incubation, Fluorescence, Infection, Inhibition, Activity Assay, Purification, Concentration Assay

Journal: bioRxiv

Article Title: Robust neutralization assay based on SARS-CoV-2 S-bearing vesicular stomatitis virus (VSV) pseudovirus and ACE2-overexpressed BHK21 cells

doi: 10.1101/2020.04.08.026948

Figure Lengend Snippet: Verify the correlation between VSVdG-SARS-CoV-2-Sdel18 based system and live SARS-CoV-2 system. A Analyze the neutralizing antibodies in the serum of 18 convalescent patients using VSVdG-SARS-CoV-2-Sdel18 based system. ID50 was used to indicate the neutralizing activity of sera. B Analyze the neutralizing antibodies in the serum of 18 convalescent patients using live SARS-CoV-2 system. ID50 was used to indicate the neutralizing activity of sera. C The correlation between VSVdG-SARS-CoV-2-Sdel18 based system and live SARS-CoV-2 system.

Article Snippet: SARS-CoV-2 spike specific antibody was generated by immunized Balb/c mouse with SARS-CoV-2 spike protein (RBD, Fc Tag, 40592-V05H, Sino Biological inc).

Techniques: Activity Assay