Bm condimed

To generate anti-CD169 antibody, HEK293T cells that express CD169 molecules were injected intraperitoneally three times into Wistar rats. TiterMax Gold (TiterMax, GA) was also injected at the initial immunization. Splenocytes were fused with NSO^bcl2 myeloma cells55 (link) by PEG1500 (Roche, Germany). Hybridoma cells were selected in DMEM/10% FCS containing HAT (Sigma, MO) and 1% BM-Condimed (Roche). Clone M7 that produces antibodies against the CD169 molecule stably expressed on WR19L cell line (WR-CD169 cells) was selected by flow cytometry. To generate anti-CCL8 antibody, the Wistar rat was immunized subcutaneously in the foot pad with CCL8 peptide (1–13: EKLTGPDKAPVTC) emulsified in adjuvant. Approximately 1,600 hybridomas were established by fusing lymph node cells with myeloma cells as described above. The hybridoma supernatants were tested by ELISA, and hybridomas that specifically detect recombinant CCL8 protein were selected.

To generate anti-histone H3 (citrulline R2 + R8 + R17) antibodies, a Wistar rat was subcutaneously immunized in the foot pad with a peptide derived from human histone H3 (citrulline R2 + R8 + R17) (2–20: A(cit)RTKQTA(cit)RKSTGGKAP(cit)RKQ) emulsified in adjuvant (TiterMax Gold; TiterMax). Splenocytes were fused with NSO^bcl2 myeloma cells^{61 (link)} using PEG1500 (Roche, Germany). Hybridoma cells were selected in DMEM/10% FCS containing HAT (Sigma) and 5% BM-Condimed (Roche). The hybridoma supernatants were tested using ELISA, and a monoclonal antibody designed to specifically detect human histone H3 (citrulline R2 + R8 + R17) peptides but not human non-citrullinated histone H3 peptides was obtained (11-11B-4F). The IgG fraction was purified using a Protein G affinity column (GE Healthcare), and the buffer exchange to PBS was performed using PD-10 columns (GE Healthcare). The antibody was biotinylated using EZ-Link Sulfo-NHS-LC-LC-Biotin (Thermo Fisher Scientific) according to the manufacturer’s recommendations.