Development of Monoclonal Antibody and Diagnostic Test for Middle East Respiratory Syndrome Coronavirus Using Cell-Free Synthesized Nucleocapsid Antigen

Abstract

Protein nativity is one of the most critical factors for the quality of antigens used as immunogens and the reactivities of the resultant antibodies. The preparation and purification of native viral antigens in conventional cell-based protein expression systems are often accompanied by technical hardships. These challenges are attributable mainly to protein aggregation and insolubility during expression and purification, as well as to very low expression levels associated with the toxicity of some viral proteins. Here, we describe a novel approach for the production of monoclonal antibodies (mAbs) against nucleocapsid protein (NP) of the Middle East respiratory syndrome coronavirus (MERS-CoV). Using a wheat germ cell-free protein synthesis system, we successfully prepared large amounts of MERS-CoV NP antigen in a state that was highly soluble and intact for immunization. Following mouse immunization and hybridoma generation, we selected seven hybridoma clones that produced mAbs with exclusive reactivity against MERS-CoV NP. Epitope mapping and subsequent bioinformatic analysis revealed that these mAbs recognized epitopes located within relatively highly conserved regions of the MERS-CoV amino-acid sequence. Consistently, the mAbs exhibited no obvious cross-reactivity with NPs derived from other related viruses, including SARS coronavirus. After determining the optimal combinations of these mAbs, we developed an enzyme-linked immunosorbent assay and a rapid immunochromatographic antigen detection test that can be reliably used for laboratory diagnosis of MERS-CoV. Thus, this study provides strong evidence that the wheat germ cell-free system is useful for the production of diagnostic mAbs against emerging pathogens.

Molecular Characteristics, Functions, and Related Pathogenicity of MERS-CoV Proteins

“…Reports have described new methods to generate mAbs directed against N proteins. Using the wheat germ cell protein synthesis system, a large number of MERS-CoV N protein antigens were successfully prepared in a highly soluble and intact immune state [89].…”

A novel luciferase immunosorbent assay performs better than a commercial enzyme-linked immunosorbent assay to detect MERS-CoV specific IgG in humans and animals

“…Several methods can be used to detect serum antibodies against MERS-CoV, such as enzyme-linked immunosorbent assay (ELISA), microneutralization (MN), immunofluorescence assay (IFA), the plaque reduction neutralization test (PRNT), and the pseudovirus particle neutralization test (ppNT). Among these assays, MN, IFA, and PRNT depend on virus cultivation, and thus, require equipment and facilities located within a Biosafety Level 3 (BSL-3) laboratory [1,3,12,18,35,39,[50][51][52][53][54]. While there are commercial anti-MERS-CoV serologic test kits for humans and animals including ELISA and IFA, rapid and sensitive serological assays for the detection of anti-MERS-CoV IgG independent of purified protein, specific secondary antibody, virus cultivation and BSL-3 facilities are still urgently needed.…”

Emerging infectious disease laboratory and diagnostic preparedness to accelerate vaccine development

“…62 EID public health and countermeasure programs have unique challenges for diagnostic and vaccine clinical assay development purposes. 2,39,41,[63][64][65][66][67][68][69][70][71] There may be an incomplete understanding of the biology or epidemiology of a new pathogen, which can delay or confound the selection of a relevant vaccine target and the subsequent assay development to be used to evaluate the candidates. The field may suffer from a lack of available reagent sources or with inconsistency in quantity and quality of those available, especially early in the discovery and development process.…”