Inclusion of MERS-spike protein ELISA in algorithm to determine serologic evidence of MERS-CoV infection

Abstract

The Centers for Disease Control and Prevention (CDC) algorithm for detecting presence of serum antibodies against Middle East Respiratory Syndrome coronavirus (MERS-CoV) in subjects with potential infections with the virus has included screening by indirect ELISA against recombinant nucleocapsid (N) protein and confirmation by immunofluorescent staining of infected monolayers and/or microneutralization titration. Other international groups include indirect ELISA assays using the spike (S) protein, as part of their serological determinations. In the current study, we describe development and validation of an indirect MERS-CoV S ELISA to be used as part of our serological determination for evidence of previous exposure to the virus.

Keywords: MERS-CoV; antibodies; coronaviruses; immunity; serology; surveillance.

Figure 1

A, MERS‐CoV S ELISA Optical densities (OD) of 400 normal human sera from participants without a history of MERS‐CoV infection, and one positive control serum from a patient with a history of MERS‐CoV infection. Sera were all diluted at 1:400. Each dot is one serum. The line is OD 0.2, the cutoff of the assay. B, Optical density (OD) at 1:400, 800, and 1600 used for MERS‐S ELISA, at 1:400 for the MERS‐N ELISA, and the reciprocal endpoint titer for the micorneutralization test (MNt) of each true positive (P1‐9) and true negative (N1‐9) sample. C‐E MERS‐S Two‐Graph ROC analysis of nine true positive and nine true negative sera at 1:400 C, 1:800 D, and 1:1600 E, to determine MERS‐CoV S ELISA cut‐off OD values and their association with diagnostic sensitivity (%Se) and specificity (%Sp). The sensitivity (%Se), or true positive, and specificity (%Sp), or true negatives are plotted as percentage (y axis) vs. cut‐off OD values (x axis). F, ERS‐N ELISA two‐graph ROC analysis of true positive and true negative at 1:400

Figure 2

A, Sera from patients with known histories of human coronavirus infections 229E, NL63, OC43, HKU1, and SARS‐CoV, negative control normal human sera (NHS), and MERS‐CoV sera were diluted at 1:100, 1:400, 1:1600, and 1:6400 and used in S ELISA. The line represents the assay OD cutoff of 0.2. B, Western blot analysis of sera from patients with known histories of coronavirus infections (indicated at the bottom of the blots) against purified recombinant MERS‐CoV N, pET lysate negative control, and recombinant MERS‐CoV S