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96 well EIA/RIA plates (Costar, Corning NY) were coated with MCMV-infected MEF cell lysate, blocked with goat serum, and incubated with 2-fold serial dilutions of B6 MCMV− sera, MCMV+ sera, or transplant sera (D−/R−, D+/R−, D+/R+). Plates were developed using HRP-conjugated goat anti-mouse IgG antibodies and TMB substrate solution (Pierce Biotechnology, Rockford IL). The optical density was measured with an ELX808 Ultra Microplate Reader (BioTek Instruments, Winooski, VT) at 450 nm wavelength.
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