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.