Design and optimization of an IgG human ELISA assay reactive to recombinant RBD SARS-CoV-2 protein.
Appl Microbiol Biotechnol
; 106(23): 7933-7948, 2022 Dec.
Article
in English
| MEDLINE | ID: covidwho-2103864
ABSTRACT
Serology assays are essential tools to mitigate the effect of COVID-19, help to identify previous SARS-CoV-2 infections or vaccination, and provide data for surveillance and epidemiologic studies. In this study, we report the production and purification process of the receptor-binding domain (RBD) of SARS-CoV-2 in HEK293 cells, which allowed the design, optimization, and validation of an indirect ELISA (iELISA) for the detection of human anti-RBD antibodies. To find the optimal conditions of this iELISA, a multivariate strategy was performed throughout design of experiments (DoE) and response surface methodology (RSM), one of the main tools of quality by design (QbD) approach. The adoption of this strategy helped to reduce the time and cost during the method development stage and to define an optimum condition within the analyzed design region. The assay was then validated, exhibiting a sensitivity of 94.24 (86.01-98.42%; 95% CI) and a specificity of 95.96% (89.98-98.89%; 95% CI). Besides, the degree of agreement between quality results assessed using kappa's value was 0.92. Hence, this iELISA represents a high-throughput technique, simple to perform, reliable, and feasible to be scaled up to satisfy the current demands. Since RBD is proposed as the coating antigen, the intended use of this iELISA is not only the detection of previous exposure to the virus, but also the possibility of detecting protective immunity. KEY POINTS ⢠RBD was produced in 1-L bioreactor and highly purified. ⢠An iELISA assay was optimized applying QbD concepts. ⢠The validation procedure demonstrated that this iELISA is accurate and precise.
Keywords
Full text:
Available
Collection:
International databases
Database:
MEDLINE
Main subject:
SARS-CoV-2
/
COVID-19
Type of study:
Diagnostic study
/
Observational study
/
Prognostic study
Topics:
Vaccines
Limits:
Humans
Language:
English
Journal:
Appl Microbiol Biotechnol
Year:
2022
Document Type:
Article
Affiliation country:
S00253-022-12254-w
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