Blood samples were collected from the tail veins of mice per immunized group and pooled together on day 7, 14, 21, and 28 after primary immunization, respectively. Anti-rOmpB-4 IgGs were detected by enzyme-linked immunosorbent assay (ELISA). Briefly, 96-well plate (Nunc, Shanghai, China) was coated with 1.5 μg/ml rOmpB-4 overnight and incubated with mouse sera at the dilution of 1:1000. Then, the IgG, IgG1, or IgG2a to rOmpB-4 was determined with goat anti-mouse IgG, IgG1, or IgG2a HRP-conjugated antibodies (1:5000) and a TMB substrate kit (eBioscience, San Diego, CA) according to previous methods [32 (link)]. Absorbance at 450nm was analysed with a UVM 340 microplate reader (Asys Hitech GmbH, Eugendorf, Austria). Anti-C. burnetii phase I/II IgGs were detected by indirect immunofluorescence assay (IFA) as per methods described previously [37 (link)]. The phase I or II C. burnetii-coated slide was incubated with sera from mice immunized with rOmpB-4 mixed with CMR at two-fold dilution (initial at the dilution of 1:100) in PBS for 45 min at 37°C. After three washes with PBS, the C. burnetii cells on the slides were incubated with a 1:100 dilution of FITC-conjugated goat anti-mouse IgGs (eBioscience, San Diego, CA) for 45 min at 37°C. After another three washes, the coxiella cells on the slides were observed under a fluorescence microscope (Olympus BX60).
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