For PRN assays, the Chicago-1 strain of MeV was mixed with serially diluted plasma and assayed for plaque formation on Vero cells in triplicate. The dilution of plasma that resulted in 50% plaque reduction was calculated. MeV-specific IgG and IgA and H-specific IgG in plasma were measured by EIAs using MaxiSorp plates (Nunc) coated with lysates from MeV-infected Vero cells (1.16 μg of protein per well; Advanced Biotechnologies) or MeV H-expressing L cells (118 (link)) as previously described (15 (link)). Avidity of MeV-specific IgG was determined by adding increasing concentrations of ammonium thiocyanate (NH4SCN; 0.5 to 3 M) to the EIA assay for 15 min. The avidity index was calculated as the concentration of NH4SCN at which 50% of the bound antibody was eluted.
To measure antibody-secreting cells in the bone marrow, cells isolated from bone marrow aspirates by density gradient centrifugation using Lympholyte Mammal (Cedarlane Laboratories) were incubated for 6 hours with Multiscreen ELISpot plates (MilliporeSigma) coated with MeV-infected Vero cell lysate, H-expressing L cell lysate, or baculovirus-expressed N. Bound immunoglobulin was detected with horseradish peroxidase (HRP)–conjugated goat anti-monkey IgG (Nordic), developed with stable diaminobenzidine (DAB) solution and read on an ImmunoSpot plate reader (Cellular Technology).