Development and validation of different indirect ELISAs for MERS-CoV serological testing

Abstract

Since 2012, MERS-CoV has caused up to 2220 cases and 790 deaths in 27 countries with Saudi Arabia being the most affected country with ~83.1% of the cases and ~38.8% local death rate. Current serological assays such as microneutralization (MN), plaque reduction neutralization, immunofluorescence, protein microarray or pseudoparticle neutralization assays rely on handling of live MERS-CoV in high containment laboratories or need for expensive and special equipment and reagents and highly trained personnel which represent a technical hurdle for most laboratories in resource-limited MERS-CoV endemic countries. Here, we developed, compared and evaluated three different indirect ELISAs based on MERS-CoV nucleocapsid protein (N), spike (S) ectodomain (amino acids 1-1297) and S1 subunit (amino acids 1-725) and compared them with MN assay. The developed ELISAs were evaluated using large number of confirmed seropositive (79 samples) and seronegative (274 samples) MERS-CoV human serum samples. Both rS1- and rS-ELISAs maintained high sensitivity and specificity (≥90%) across a wider range of OD values compared to rN-ELISA. Moreover, rS1- and rS-based ELISAs showed better agreement and correlation with MN assay in contrast to rN-ELISA. Collectively, our data demonstrate that rS1-ELISA and rS-ELISA are more reliable than rN-ELISA and represent a suitable choice for seroepidemiological testing and surveillance in MERS-CoV endemic regions.

Keywords: ELISA; MERS-CoV; Saudi Arabia; Serology.

Fig. 1

MERS-CoV recombinant proteins. (A) Recombinant MERS-CoV proteins were detected by Western blot using (A) anti-His tag Abs, (B) known seropositive human serum sample or (C) known seronegative human serum sample. All experiment showed protein bands with expected sizes.

Fig. 2

Cut-off values for the developed ELISAs. A 100 serum samples serologically negative for MERS-CoV from healthy blood donors were used to determine the cut-off values for (A) rN-ELISA, (B) rS1-ELISA and (C) rS-ELISA. Cut-off values were calculated as mean + 3SD. The empty square is a serologically positive control sample. The dotted line represents the cut-off of the assay.

Fig. 3

ELISA results of the serologically negative and positive serum samples. The test results for (A) rN-ELISA, (B) rS1-ELISA and (C) rS-ELISA were plotted as OD value at 650 nm for each sample group based on their MN₁₀₀ titer. The dotted line represents the cut-off of each assay.

Fig. 4

Relative sensitivity and specificity of the developed ELISAs using Two-Graph-ROC analysis. Sensitivity and specificity of (A) rN-ELISA, (B) rS1-ELISA and (C) rS-ELISA are plotted (y-axis) against OD values (x-axis).

Fig. 5

Receiver operating characteristics (ROC) analysis. ROC analysis was applied to serologically positive vs. negative MERS-CoV samples identified by MN assay at MN₁₀₀ titer of 1:10 for rN-ELISA, rS1-ELISA and rS-ELISA.