α2 6 sialyltransferase

Monoclonal ACPA from the clones 109 and C7 and anti-TNP antibodies were generated as described elsewhere30 (link)39 (link). For galactosylation, 1 mg of IgG was incubated with 10 μM UDP-galactose (Calbiochem) and 2,5 mU of β1-4 galactosyltransferase (Sigma) in 50 mM MOPS, pH7.2 with 20 mM MnCl₂ for 48 h at 37 °C. For sialylation, 1 mg of IgG was incubated with 10 μM CMP-sialic acid (Calbiochem) and 10 mU of α2-6 sialyltransferase (Sigma) in 50 mM MES, pH 6,0 with 10 mM MnCl₂ for 48 h at 37 °C. The reactions were confirmed with a lectin blot.

mBSA (2 mg ml⁻¹; Sigma) was emulsified in equal amounts with complete Freunds adjuvant (Sigma) containing 5 mg ml⁻¹ heat-inactivated Mycobacterium tuberculosis (H37Ra; Difco). Male 6-week-old Balb/c mice (Janvier) were injected s.c. with 100 μl of this emulsion and simultaneously injected i.p. with 5 × 10⁸ heat-inactivated Bordtella pertussis (National Institute for Biological Standards and Control (NIBSC)) at day 0 and 7. After 21 days, the mice were challenged with 100 μg mBSA into one knee joint. At day 28, blood was taken and IgG was isolated with a protein G column (GE Healthcare) according to the manufacturer’s instructions. For sialylation, 1 mg of IgG was incubated with 750 μM CMP-sialic acid (Calbiochem) and 30 mU of α2-6 sialyltransferase (Sigma) in 100 mM Tris/HCl, pH 8,0 with 1 mM MnCl₂ for 4 days at 37 °C. The reaction was confirmed with a lectin blot. For the IgG transfer, 2 mg of untreated or sialylated AIA-IgG was injected i.v. into naive male 9-week-old Balb/c (Janvier) mice. After 1 and 5 days, the right knee joints were injected with 100 μg mBSA. The left knee joints served as internal controls. Knee joint swelling was determined using a dial thickness gauge (Peacock) and expressed relative to the knee diameter at day 0. At day 8, mice were killed and the bones were dissected for histological analysis.