Lectin Reactivity of Anti-TF IgG

The lectin reactivity of the TF-specific IgG was measured by ELISA in a similar way, except that the binding of neuraminic acid- (sialic acid-) specific Sambucus nigra agglutinin (SNA) and mannose-specific concanavalin A (ConA) to the absorbed serum anti-TF Abs (all isotypes) or anti-TF IgG from tIgG samples was measured as described elsewhere [19 (link), 21 (link)]. Biotinylated SNA (Vector Laboratories Inc., USA) in 10 mmol/L HEPES, 0.15 mol/L NaCl, and 0.1 mmol/L CaCl₂, pH 7.5, and biotinylated ConA (Sigma, USA) in the ConA binding buffer (0.05 mol/L Tris-HCl buffer, pH 7.2, containing 0.2 mol/L NaCl and 3 mmol/L CaCl₂, MgCl₂, and MnCl₂) were both applied at a concentration of 5 μg/mL each, for 1.5 h at 25°C. The bound lectins were detected with a streptavidin-alkaline phosphatase conjugate (Dako, Denmark) and p-nitrophenylphosphate (Sigma, USA). The optical density value of control wells (no sample) was subtracted from that of Ab-coated wells to determine the lectin binding. Each sample was analyzed in duplicate.