Evaluation of MERS-CoV Neutralizing Antibodies in Sera Using Live Virus Microneutralization Assay

Abstract

The microneutralization (MN) assay is a standard and important technique in virology, immunology, and epidemiology. It is a highly specific and sensitive assay for evaluating virus-specific neutralizing antibodies (nAbs) in human and animal sera. It provides the most precise answer to whether or not an individual or animal has antibodies that can neutralize or inhibit the infectivity of a specific virus strain. However, using live virus-based MN assay might require working under high containment facilities especially when dealing with high-risk pathogens such as the Middle East respiratory syndrome-coronavirus (MERS-CoV). In this chapter, we describe the isolation, amplification, and titration of MERS-CoV, as well as detailed MN assay to measure nAb levels in sera from different mammalian species.

Keywords: MERS-CoV; Microneutralization; Neutralizing antibodies.

Fig. 1

Example of cytopathic effect (CPE) observed in Vero E6 cells infected with MERS-CoV 3 days after inoculation

Fig. 2

Schematic representation of preparation of virus dilution for MERS-CoV titration by TCID₅₀. The sequential steps of plate preparation are indicated by numbered boxes. After completion, transfer 100 μL from the 96-well U-bottom plate containing diluted MERS-CoV sample to the cells in each corresponding well in the 96-well tissue culture plate using a multichannel pipette. CC is cell control wells

Fig. 3

Plate preparation for MERS-CoV microneutralization assay. The preparation steps for MERS-CoV microneutralization assay plate are indicated by numbered boxes. After completion, incubate the serum-virus mixtures for 1 h in 37 °C incubator with 5% CO₂. Then, transfer 100 μL from the U-shaped 96-well plate containing diluted MERS-CoV sample to the cells in each corresponding well in the 96-well tissue culture plate using a multichannel pipette. VC is virus control and CC is cell control wells