Lectin Reactivity of TF Glycotope Antibodies

The lectin reactivity of TF glycotope-specific antibodies was measured in a similar way, except that the binding of the neuraminic acid (sialic acid) specific Sambucus nigra agglutinin (SNA) to the absorbed anti-TF antibodies was determined as described earlier [37 (link)]. The biotinylated SNA (Vector Laboratories, Inc., USA) in 10 mmol/L Hepes, 0.15 mol/L NaCl, 0.1 mmol/L CaCl₂, pH 7.5 was applied at a concentration of 5 μg/mL for 1.5 h at 25°C. The bound lectin was detected with a streptavidin-AP conjugate (Dako, USA) and pNPP (Sigma, USA). The OD of control wells (no serum sample) was subtracted from that of Ab-coated wells to determine the lectin binding. Each sample was analysed in duplicate. The value of the SNA binding to all TF-specific Abs and the ratio of SNA binding to the level of TF-specific IgG, IgM, and IgA (SNA/Ig index) were determined.