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

Yutaro Yamaoka,Shuetsu Fukushi,Yuki Matsushima,Tomoyuki Chimuro,Akihide Ryo,Hirokazu Kimura,Shutoku Matsuyama,Makoto Takeda,Hiroyuki Kuroyama,Satoko Matsunaga

doi:10.3389/fmicb.2016.00509

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.

Highlights

Middle East respiratory syndrome coronavirus (MERS-CoV), a novel human coronavirus, was first isolated in 2012 in the Arabian Peninsula (Zaki et al, 2012)
Among the 48 clones, seven (#5, #13, #20, #25, #29, #45, and #46) were selected for further investigation based on their reactivity to MERS-nucleocapsid protein (NP) in immunoblot analysis (Figure 1C)
We investigated the detection limit of ELISA for MERS-CoV virions permeabilized by addition of Nonidet P-40 (NP-40)

Summary

Introduction

Middle East respiratory syndrome coronavirus (MERS-CoV), a novel human coronavirus, was first isolated in 2012 in the Arabian Peninsula (Zaki et al, 2012). MERS-CoV is a positive-sense, enveloped, single-stranded RNA virus of genus Betacoronavirus within subfamily Coronavirinae (de Groot et al, 2013). MERS-CoV infection often causes fever, cough, and severe pneumonia, occasionally accompanied by renal disease (Banik et al, 2015). More than 1600 laboratory-confirmed cases with high fatality rates (∼36% mortality) have been reported (World Health Organization [WHO], 2016). Because there is currently no specific antiviral drug or vaccine approved for clinical use against MERS-CoV, rapid diagnostic tests are urgently required to manage and control this virus. Rapid and specific diagnosis is essential for preventing the spread of any kind of infectious disease

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Conclusion