Measuring Anti-TF and Anti-α-Gal IgG

The anti-TF and anti-αGal IgG antibody levels were determined by enzyme-linked immunosorbent assay (ELISA) as described elsewhere [19 (link), 21 (link)] with minor modifications. Briefly, the plates (Maxisorp, Nunc, Denmark) were coated with a synthetic TFα- or αGal-polyacrylamide conjugate (Lectinity, Russia) in carbonate buffer, pH 9.6, 5 μg per well. After overnight incubation at +4°C, triple washing, and blocking with Superblock solution (Pierce, USA) for 15 min at 25°C, the serum or purified IgG samples diluted to 1 : 25 in PBS-0.05% Tween (Tw) were applied for 1.5 hr at 25°C. The concentration of IgG in serum and tIgG samples was measured by the Easy-Titer IgG Assay Kit (Thermo Scientific, USA) and the IgG concentration in the tIgG probe adjusted to that in serum. After subsequent washing with PBS-Tw, the bound anti-TF or anti-αGal IgG was detected with alkaline phosphatase conjugated goat anti-human IgG (Dako, Denmark) and p-nitrophenylphosphate disodium hexahydrate (Sigma, USA). The absorbance values were read at 405 nm (Tecan Reader, Austria).