VLP-ELISAs were performed as previously described (29 (link)). Briefly, 96well PolySorb ELISA plates (Thermo Fisher Scientific, Rockford, IL, USA) were coated with 100 ng/well purified high quality MnPV L1-VLPs in 50 mM carbonate buffer pH9.6. The next day, plates were blocked with CBB and incubated for 1 h with three-fold serial dilutions (ranging from 1:100 to 1:656,100) of Mastomys sera in CBB. Then, plates were washed four times with PBST and incubated with goat anti-mouse IgG-HRP (1:10,000 in CBB, Promega GmbH, Walldorf, Germany). After four washes, color development and measurement was performed as described for the GST-ELISA. Antibody titer represents the last reciprocal serum dilution above the blank. The cut-off was set to a titer of 300 based on previous experiences with measuring sera of animals from virus-free and naturally MnPV-infected colonies (28 (link)).
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