Design, optimization and validation of a RBD indirect ELISA for detecting antibodies against SARS-CoV-2

RODRÍGUEZ, MARÍA CELESTE; CEAGLIO, NATALIA; FUSELLI, ANTONELA; VILLARRAZA, CARLOS JAVIER; GARAY, ERNESTO; ANTUÑA, SEBASTÍAN; GASTALDI, VICTORIA; FONTANA, DIEGO; GUGLIOTTA, AGUSTINA; PRIETO, CLAUDIO

Santa Fe

Simposio; IX Simposio Latinoamericano de Tecnología de Cultivos Celulares - IX SLATCC 2022; 2022

Centro Biotecnológico del Litoral - FBCB - UNL

Testing strategies are essential tools to mitigate the effect of COVID-19, help to diagnose the disease and provide data for surveillance and epidemiologic studies. Virus neutralization tests (VNTs) are the gold standard of serology assays. However, they exhibit several drawbacks, including the requirement of containment facilities and the difficulty of standardization. In this context, serological binding tests based on antigen-antibody interaction represent a more versatile alternative.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, validation and optimization of an indirect ELISA (iELISA) for the detection of human anti-RBD antibodies. To find the optimal conditions of this iELISA, an optimization procedure through design of experiments (DoE) and response surface methodology (RSM) approach was performed, applying a full factorial design at three levels. Thereafter, an optimal condition was found: 136.36 ng of RBD as coating antigen and 1/3225 of commercial HRP-conjugated anti-human IgG for detection (Santa Cruz Biotechnology). Then, the assay was 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 of the region. 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 possibly of detecting protective immunity.