Cost-effective serological test to determine exposure to SARS-CoV-2: ELISA based on the receptor-binding domain of the spike protein (Spike-RBD_N318-V510) expressed in Escherichia coli

Abstract

Massive worldwide serological testing for SARS-CoV-2 is needed to determine the extent of virus exposure in a particular region, the ratio of symptomatic to asymptomatic infected persons, and the duration and extent of immunity after infection. To achieve this aim, the development and production of reliable and cost-effective SARS-CoV-2 antigens is critical. Here, we report the bacterial production of the peptide S-RBDN318-V510, which contains the receptor binding domain of the SARS-CoV-2 spike protein. We purified this peptide using a straightforward approach involving bacterial lysis, his-tag mediated affinity chromatography, and imidazole-assisted refolding. The antigen performances of S RBDN318 V510 and a commercial full-length spike protein were compared in two distinct ELISAs. In direct ELISAs, where the antigen was directly bound to the ELISA surface, both antigens discriminated sera from non-exposed and exposed individuals. However, the discriminating resolution was better in ELISAs that used the full-spike antigen than the S-RBDN318-V510. Attachment of the antigens to the ELISA surface using a layer of anti-histidine antibodies gave equivalent resolution for both S-RBDN318-V510 and the full length spike protein. Our results demonstrate that ELISA-functional SARS-CoV-2 antigens can be produced in bacterial cultures. S-RBDN318-V510 is amenable to massive production and may represent a cost-effective alternative to the use of structurally more complex antigens in serological COVID-19 testing.

Expression of SARS-CoV-2 surface glycoprotein fragment 319–640 in E. coli, and its refolding and purification

“…The procedure for purification of SARS-CoV-2 Spike fragment 319-640 from inclusion bodies in E. coli described here can be applied to any Spike fragment. Recently, S318-510 fragment was expressed in E. coli and successfully used in ELISA assays 16 . Our work suggests that this technique can be applied to longer Spike fragments, including those that are difficult to express in mammalian cells.…”

Purification and characterization of the receptor‐binding domain of SARS‐CoV‐2 spike protein from Escherichia coli

“…Some studies suggest that RBD of SARS‐CoV S protein expressed by E. coli could provide protective immunity [26,27]. Moreover, RBD (N318‐V510) of SARS‐CoV‐2 S protein was expressed by E. coli and has been used as a cost‐effective antigen for worldwide serological testing [28]. However, RBD of SARS‐CoV‐2 expressed in E. coli (RBD‐1) lacks the disulfide bond formation and glycosylation.…”

Automated ELISA on-chip for the detection of anti-SARS-CoV-2 antibodies

“…Under the current circumstances, where millions of viral infections exist, is un-deniable that vaccination is of vital importance, and immunoassays have taken a particularly relevant role in identifying and monitoring patients' immune responses over time. Therefore, several research groups have proposed various COVID-19-related immunoassays which are mainly based on viral antigens, such as the spike protein [13], receptor-binding domain (RBD) [14], and nucleoprotein [15] and include different strategies ranging from the traditional enzyme-linked immunoassay (ELISA) to more complex microfluidic immunoassays [16][17][18]. Through the course of the COVID-19 pandemic, several diagnostic challenges have emerged, particularly regarding the millions of tests require to face the disease spreading.…”