Legends
Figure 1 . SARS-CoV-2 S protein including the ectodomain
(residues 19-1207) without native signal peptide. The furin cleavage
site was removed (GSAS mutations) and residues at positions 986 (K) and
987(V) were mutated to proline. The C-terminal included a 4 fibritin
trimerization domain, a TEV protease cleavage site, and a histidine tag.
Figure 2 . Rachiplusia nu larvae. a. A batch of 500
fifth-instar larvae. b. Injection of the recombinant baculovirus near
the third prolegs.
Figure 3. Purification of S protein by IMAC. a. SDS-PAGE with
Coomassie blue staining. b. Western blot with DAB staining. Lanes: MK.
molecular weight marker. 1. Larval extract (input). 2. Passthrough. 3.
Wash fraction with 80 mM imidazole. 4. Elution fraction with 500 mM
imidazole.
Figure 4. Size exclusion analysis of S protein purified by
IMAC. a) Chromatogram showing the elution profile of S protein in IMAC.
The void volume (Blue dextran) of the column is ~8.8 ml,
and the monomeric (~67 kDa) and dimeric
(~140 kDa) forms of Bovine Serum Albumin (BSA) eluting
at ~12.5 ml and ~14.4 ml, respectively,
are shown for reference with bars at the top of the chromatogram. The
asterisk indicates the peak containing Spike trimer. Fractions subjected
to WB staining with ECL (from 2 to 14) are indicated at the bottom of
the chromatogram. 4b) WB of SEC eluted fractions. Protein was revealed
using an anti 6xHis tag antibody and ECL. Lane M, molecular weight
markers; lane C, positive control; lane 1, input; lanes 2 to 14
correspond to respective fractions obtained from the SEC column.
Figure 5 : Analysis of ELISA performance resulting from the
study of 82 sera from normal control individuals and 99 sera from
COVID-19 patients. (A) Sensitivity curve (o) and specificity (x) as a
function of the possible cut-off values. The vertical dashed line
indicates the cut-off value with the optimized sensitivity and
specificity parameters (cut-off = 3.0). (B) ROC curve analysis of ELISA,
AUC is included.
Figure 6 : Antibodies anti-SARS-Cov 2 results obtained by ELISA
in sera from normal control individuals and sera from COVID 19 patients
IgG positive and negative by COVIDAR IgG ELISA test. Results are
expressed as SDs. The cut-off value (SDs >3.0) is indicated
by a dotted line and medians for each population are indicated as a full
line (***p<0.0001).