Cytomegalovirus Seroprevalence Assessment via ELISA

As described by Kilgour et al, CMV ELISA testing (University of Birmingham, Birmingham UK) was used to ascertain participant’s CMV status. Briefly, mock and viral lysate were used to coat 95 well plate overnight at 4 °C. Using plasma from 3 CMV positive donors, a standard was prepared and added to the plate in a 1 in 4 serial dilution, alongside 1ul of healthy donor samples. After 1 h incubation at room temperature, the plate was washed and anti-IgG horseradish peroxidase conjugated secondary antibody (Southern Biotech, Alabama, USA) added to each well and incubated for a further 1 h in the dark, RT. After repeat wash steps, 100 μL of TMB ELISA peroxidase substrate (Rockland Immunochemicals, Pennsylvania, USA) was added and incubated for 10 min in the dark, RT. To stop the reaction, 100 μL of 1 mM HCl was added, prior to reading on an ELISA plate reader at 450 nm [44 (link)].