Optimization and validation of a simple and reliable ELISA assay based on recombinant RBD domain to detect human anti-SARS-CoV-2 antibodies

RODRÍGUEZ, MARÍA CELESTE; CEAGLIO, NATALIA; GUGLIOTTA, AGUSTINA; VILLARRAZA, CARLOS JAVIER; GARAY, ERNESTO; FUSELLI, ANTONELA; GASTALDI, VICTORIA; TARDIVO, MARÍA BELÉN; ANTUÑA, SEBASTIÁN; PRIETO, CLAUDIO

Viena

Encuentro; ESACT Frontiers Retreat; 2022

European Society of Animal Cell Technology

BACKGROUND AND NOVELTYOn 11th March 2020 the WHO declared the novel coronavirus outbreak a pandemic, and since then a global effort to mitigate the impact of COVID-19 disease has been done. In this context, testing strategies can help to diagnose the infection, estimate de durability of vaccine-mediated immunity and the optimal timings of booster vaccinations. 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. Hence, serological 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 optimization and validation of an indirect ELISA (iELISA). To our knowledge, this is the first ELISA for detecting human anti-RBD antibodies in which Quality by Design (QbD) tools have been applied during the optimization stage. Interestingly, since this iELISA can detect antibodies against RBD, it can be proposed as a technique to assess protective immunity.EXPERIMENTAL APPROACHTo obtain the recombinant version of RBD, expression plasmids were used for transient transfections of sHEK293 cells under adherent conditions. After six rounds of successive transfection and puromycin selection, the sHEK cell line that exhibited the highest productivity was gradually adapted to a chemically defined serum-free medium. Afterwards, cells were transferred to a one-liter bioreactor operated in perfusion mode. RBD protein was purified from clarified culture supernatants by immobilized metal affinity chromatography (IMAC). To better characterize RBD, purified samples were analyzed by RP-HPLC, fluorescent spectroscopy and SDS-PAGE after PNGase F treatment. To find the optimal conditions of the iELISA, a multivariate strategy was performed throughout design of experiments (DoE) and response surface methodology (RSM). A three-level full factorial design was performed, with two factors [amount of RBD and dilution of HRP-conjugated anti-human IgG (Santa Cruz Biotechnology)] and four center points. Thereafter, to assess the reliability of the developed iELISA, a validation procedure was performed.RESULTS AND DISCUSSIONWe expressed, produced and highly purified the RBD domain in suspension HEK293 cells. After a single purification step, the RBD was recovered with a very high purity level (>95%), which is critical for obtaining reliable results in the development of serology assays. To have a serological assay that can demonstrate possible evidence of acquired immunity, we optimized and validated an iELISA for the detection of human anti-RBD antibodies. Here, the optimization was achieved applying DoE. The adoption of this strategy helps us to reduce the time and cost during the method development stage and define an optimum condition within the analyzed design region (136.36 ng of RBD and 1/3225 of HRP-conjugated anti-human IgG). 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. Furthermore, assay repeatability and intermediate precision rendered acceptable CV% values. Hence, this iELISA represents a high-throughput technique, simple to perform, accurate, precise and feasible to be scaled-up.