Np ficoll

16–30-wk-old mice were i.p. immunized with 50 μg of NP-Ficoll (Biosearch Technologies) in 200 μl. For VLP immunization, mice 12–18 wk of age were injected i.p. with 2 μg/250 μl VLPs or 250 μl 1× PBS. VLPs were provided by B. Hou (Chinese Academy of Sciences, Beijing, China). For PPV23 (Merck) injection, mice were injected with 0.125 μg/200 μl i.p. per mouse or 200 μl 1× PBS.

Mice were injected with 50 µg NP(36)-KLH (Biosearch Technologies) in PBS + 50 µl Adjuvans Imject Alum (Thermo Fisher Scientific) in a 1:1 dilution i.p. for primary immunization and with 10 µg NP(36)-KLH i.p. for boost immunization. Alternatively, mice were challenged with 50 µg NP-Ficoll (Biosearch Technologies) or with ∼10⁹ SRBCs (Fiebig Nährstofftechnik, Idstein-Niederauroff/Ts.) i.p. in PBS.
For detection of NP-specific antibodies, microtiter plates were coated overnight with NP-BSA IgG1 at 4°C and blocked with PBS/2% FCS. Sera were diluted in PBS/2% FCS, starting with a 1/3,000 dilution for IgG or 1/1,000 for IgM and IgG1, plated in eightfold serial dilutions and incubated for 1 h at room temperature. HRP-conjugated IgM/IgG and IgG1 detection antibodies were obtained from SouthernBiotech. For basal Ig, Elisa microtiter plates were coated with 1 µg/ml Ig purified unlabeled antibodies (SouthernBiotech).
For quantification of anti-dsDNA antibody titers, microtiter plates were precoated with 50 µl poly-l-lysine in Tris EDTA (TE) buffer, pH 8, overnight at 4°C, washed three times with TE buffer, and coated with calf thymus dsDNA in TE buffer over night with the following detection procedure as described previously. All microtiter plates were analyzed at 490 nm on a SpectraMax 190 Microplate Reader (Molecular Devices).