Antibody tests
MERS-CoV antibody levels in all sera collected from contacts were measured using a recombinant S1 protein-coated human anti-MERS-CoV (immunoglobulin G [IgG]) ELISA kit (Euroimmun, Luebeck, Germany), which was used according to the manufacturer’s protocol. The results of the tested samples were determined by calculating the ratio of the optical density (OD) value of the sample to the OD value of the calibrator. Ratios ≥ 1.1 were considered positive, ratios ≥ 0.8 to < 1.1 were considered borderline, and ratios < 0.8 were considered negative.
IFA slides were coated with MERS-CoV non-infected or infected Vero cells. Serum samples were diluted in phosphate-buffered saline (PBS) to 1:10, 1:100, 1:250, and 1:1,000, transferred to IFA slides, and incubated for 30 minutes at 37°C in a humidified chamber. The IFA slides were washed 3 times in PBS-T for 5 minutes each and incubated with fluorescein isothiocyanate-conjugated rabbit anti-human IgG (Abcam, Cambridge, MA, USA) diluted at 1:800 for 30 minutes at 37°C in a humidified chamber. After washing 3 times in PBS-T for 5 minutes, the slides were embedded with a mounting fluid, topped with a cover glass, and observed under a fluorescent microscope.
The neutralizing antibodies in the serum samples were measured by PRNT. The PRNT procedures were performed as follows. In brief, 1:10 diluted sera were heat-inactivated at 56°C for 30 minutes. Heat-inactivated sera were diluted serially 4-fold. After an equal volume of virus (MERS-CoV/KOR/KNIH/002_05_2015) was added to the volume of serum dilutions, these mixtures were incubated at 37°C for 2 hours. The mixtures were added to each well of the 24-well plate cultured with Vero cells. The plate was incubated at 37°C for 60 minutes, and 1 mL of 1.5% carboxymethylcellulose overlay medium was then added. After incubation for 3-4 days, cell staining was performed by crystal violet. The titer of neutralizing antibody by PRNT50 was calculated using the Kärber formula, as described previously [22]. A titer above 1:20 was interpreted as positive. All sera with a positive or borderline reaction in ELISA were tested by the IFA and PRNT assays for confirmation, and cases with positive results from any 2 assays were considered to be anti-MERS positive.
This study received approval from the bioethics committee of the KCDC (2015-08-EXP-03-P-A) and the institutional review board of the National Cancer Center (NCC 2016-0058). Informed consent was obtained from study participants or their parent or legal guardian for children under 14.