Development of a diagnostic system for detection of specific antibodies and antigens against Middle East respiratory syndrome coronavirus

Abstract

Middle East respiratory syndrome coronavirus (MERS-CoV) is a single-stranded RNA virus that causes severe respiratory disease in humans with a high fatality rate. Binding of the receptor binding domain (RBD) of the spike (S) glycoprotein to dipeptidyl peptidase 4 is the critical step in MERS-CoV infection of a host cell. No vaccines or clinically applicable treatments are currently available for MERS-CoV. Therefore, rapid diagnosis is important for improving patient outcomes through prompt treatment and protection against viral outbreaks. In this study, the aim was to establish two ELISA systems for detecting antigens and antibodies against MERS-CoV. Using a recombinant full-length S protein, an indirect ELISA was developed and found to detect MERS-CoV-specific antibodies in animal sera and sera of patient with MERS. Moreover, MAbs were induced with the recombinant S protein and RBD and used for sandwich ELISA to detect the MERS-CoV S protein. Neither ELISA system exhibited significant intra-assay or inter-assay variation, indicating good reproducibility. Moreover, the inter-day precision and sensitivity were adequate for use as a diagnostic kit. Thus, these ELISAs can be used clinically to diagnose MERS-CoV.

Keywords: ELISA; Middle East respiratory syndrome coronavirus; receptor binding domain; spike protein.

Figure 1

Characterization of MERS‐CoV S protein expressed in a baculovirus system. (a) Purified S protein was loaded onto SDS‐PAGE in duplicate lanes and stained with Coomassie blue. (b) Purified S protein was negatively stained with uranyl and examined at 67,000× magnification under an electron microscope.

Figure 2

Characterization of MERS‐CoV RBD expressed in E. coli. (a) Mouse RID‐RBD protein was expressed in the soluble fraction of IPTG‐induced E. coli. IN, IPTG induction; M, protein marker; NI, no IPTG induction; P, cell pellets; S, supernatant; T, total lysate. (b) Purified mRID‐RBD and BSA were loaded onto SDS‐polyacrylamide gels in each lane.

Figure 3

Detection of MERS‐CoV antibodies in animal sera with S protein. Purified S protein was used to coat the plate and ELISA performed to identify antibodies against MERS‐CoV in rat and rabbit sera. (a) Sera of rats immunized with S protein were serially diluted and tested. (b) Sera of rabbits immunized with heat‐inactivated MERS‐CoV were analyzed under the same conditions. NC, negative control.

Figure 4

Detection with S protein of MERS‐CoV antibodies in sera of patients with MERS. MERS‐CoV‐specific antibodies in sera of patients with MERS from two hospitals were measured by ELISA using S proteins as coating antigens. (a) Patient sera from one hospital were serially diluted, and antibody titers against MERS‐CoV S protein measured. (b) Patient sera from the other hospital were analyzed under the same conditions except for use of different dilutions. [Color figure can be viewed at http://wileyonlinelibrary.com]

Figure 5

Determination of detector and capture antibodies for MERS‐CoV ELISA. (a) Schematic representation of sandwich ELISA method for the pairwise MAb tests. (b) Distribution of values using #16 MAb as the capture antibody and #89 MAb as the detector with 100 ng of S protein as the antigen (Ag). Results are shown as OD₄₅₀. (c) Distribution of values using #9 MAb as the capture antibody and #16D4 MAb as the detector. ELISA conditions were the same as above. (d) Two combinations (#16/#89 and #9/#16D4) were tested to identify the optimal MAb pair. ELISA was performed using serially diluted S protein. [Color figure can be viewed at http://wileyonlinelibrary.com]

Figure 6

Validation of MERS‐CoV antigen ELISA. MERS‐CoV antigen ELISA was tested with various S protein dilutions to confirm linearity and reproducibility. MAbs #9 and #16D4 were used as capture and detector antibodies, respectively. (a) Intra‐assay test results showing variations between wells (n = 12). (b) Inter‐assay test results showing variations between plates (n = 12). (c) Inter‐day precision test results showing variations between assays conducted at different time points. Black: Day 0, red: Day 7. (d) The LLD was determined by adding 3 × SD of the blank to the average of the blank (n = 12). OD values were used in the standard curve formula to calculate the minimum detectable concentration. [Color figure can be viewed at http://wileyonlinelibrary.com]

Figure 7

Validation of MERS‐CoV antibody ELISA. MERS‐CoV antibody ELISA was tested with various MERS‐CoV‐specific MAb dilutions to confirm linearity and reproducibility. S protein was used as a coating antigen. (a) Intra‐assay test results showing variations between wells (n = 12). (b) Inter‐assay test results showing variations between plates (n = 12). (c) Inter‐day precision test results showing variations between assays conducted at different time points. Black: Day 0, red: Day 7. (d) Lower limit of detection (LLD) was calculated by adding 3 × the standard deviation of the blank to the average of the blank (n = 12). OD values were used in the standard curve formula to calculate the minimum detectable concentration. [Color figure can be viewed at http://wileyonlinelibrary.com]