SPIKE application in an immunoassay for antibodies for
SARS-CoV-2 assessment
The immunochemical behavior of the recombinant version of the S protein
expressed in insect larvae R. nu was evaluated during the
following use as an immobilized antigen in the development of an
indirect ELISA aimed at detecting the presence of anti-S specific IgG
antibodies in human serum/plasma samples. After an initial round of
optimization (antigen concentration and binding conditions, primary and
secondary antibodies dilution, washing steps), a panel of 82 human
control serum/plasma and 99 COVID-19 patients’ serum/plasma were
analyzed. To calculate the coefficient variation, a positive serum from
a COVID-19 patient was employed. The intra-assay coefficient variation
was 3.21% (n = 2) and the inter-assay coefficient variation was 20.11%
(n = 4). The test performance was optimized in terms of sensitivity and
specificity by evaluating the effect of different cut-off values (in
SDs) on receiver operating characteristic curves (ROC) (Figure 5). The
area under the ROC curve (AUC) was 0.883, indicating that the method had
high accuracy to distinguish between samples from the two groups under
study (Carter et al., 2016). When a cut-off value of 3.0 SDs was
established, 76 out of 79 COVID-19 samples that tested positive by
COVIDAR IgG ELISA Test, were also positive by our developed ELISA
(sensitivity: 96.2%). The specificity, calculated as 100% minus the
percentage of true negative samples (normal human sera, n = 82) detected
as positive, was 98.8%. The median SDs range of true negative samples
was -0.14 (range: -1.78 to 3.21) and the median SDs range of true
positive samples was 7.32 (range: -1.31 to 23.20). Moreover, the
concordance between the ELISA developed herein and the COVIDAR IgG ELISA
test was 95.96%, with a kappa statistic of 0.879, representing a
substantial agreement between the two methods. The results presented in
the present work demonstrate that the Spike protein produced in insect
larvae is immunoreactive against the sera from COVID-19 patients and can
be used in immunoassays for the detection of anti-SARS-Cov-2 antibodies
(Figure 6). The strategy reported herein should be accessible to many
laboratories and should allow the easy production of Spike. In turn, the
availability of properly folded Spike would encourage researchers to
improve current and develop new immunochemical tests for anti-SARS-Cov-2
antibodies detection.