Np3 bsa

For immunization, mice were injected intra-peritoneally with 50 μg NP₂₃-KLH (Biosearch Technology) in 4 mg Alum (ThermoScientific). Blood samples were taken from the lateral tail-vein on day 0, 3, 7, 13, 28 after immunization. For infection, 10⁴ PFU of Vaccinia Virus Western Reserve strain (vacv) was injected into isoflurane-anesthetized animals in the footpads. NP-specific antibody titers were detected by ELISA, using NP₂₃-BSA, NP₃-BSA, and biotinylated anti–mouse IgM and IgG (Southern Biotech). Titers were determined from the dilution curve in the linear range of absorbance. All non-commercial ELISA plates were developed with alkaline-phosphatase streptavidin (Sigma) and phosphorylated nitrophenyl-phosphate (Sigma). Absorbance at 405 nm was determined with a SPECTRAmax190 plate reader (Molecular Devices). NP-specific antibody-secreting cells and vacv-specific antibody-secreting cells were captured using activated ELISPOT plates coated with either NP₂₃-BSA or vacv respectively. Detection was made using biotinylated anti–mouse IgM (Southern Biotech), IgG (Southern Biotech). All ELISPOT plates were developed with alkaline-phosphatase streptavidin (Sigma) and the BCiP®/NBT reaction (Sigma). Images were acquired using CTL 4.0 (ImmunoSpot®).

For T-independent immunisation, mice were injected intraperitoneally with 2 μg of NP-LPS (Biosearch Technologies). Blood samples were taken from the lateral tail vein on days –1, 3, 6, and 9. For T-dependent immunisation, the mice were injected intraperitoneally with 50 μg NP₂₃-KLH (Biosearch Technologies) in 4 mg alum (Thermo Fisher Scientific). Blood samples were taken from the lateral tail vein on days –1, 3, 7, 14, 21, 28, and 69. In both cases, NP-specific antibody titres were detected by ELISA, using NP₂₃-BSA, NP₃-BSA (for T-dependent immunisation), and biotinylated anti-mouse IgM and IgG (Southern Biotech). Titres were determined from the dilution curve in the linear range of absorbance. All noncommercial ELISA plates were developed with alkaline-phosphatase streptavidin (Sigma-Aldrich) and phosphorylated nitrophenyl-phosphate (Sigma-Aldrich). Absorbance at 405 nm was determined with a SPECTRAmas190 plate reader (Molecular Devices).