2.2.4. Determination of ACIVAbs IgM

To measure ACIVAbs IgM concentrations, a sandwich ELISA was used. The assay was performed as follows: a microtiter 96-well polystyrene plate was coated with 100 μL of 10 μg/mL of human CIV (Sigma-Aldrich, St. Louis, MO, USA) at room temperature for 3 h, followed by overnight incubation at 4 ◦C. The plate was washed with phosphate-buffered saline (PBS) containing 0.05% Tween 20 and 1% bovine serum albumin (BSA; Cat. No. A2153, Sigma-Aldrich, St. Louis, MO, USA). Then, a 100-μL serum sample (diluted 1:10) was placed in each well of a microtiter plate and incubated for 1 h at 37 ◦C. After washing three times, 100 μL of goat anti-human IgM Ab, Fc5μ, HRP conjugate (AP114P, Sigma-Aldrich, St. Louis, MO, USA) were added to each well for 1 h at 37 ◦C. All immunoconjugates were diluted 1:10,000 with PBS containing 1% BSA and 0.05% Tween 20. The plate was incubated for 1 h at 37 ◦C. Ortho-phenylenediamine (4 mg/mL in 0.05 M citrate buffer, pH 5.0 with H2O2) was used as a colorimetric substrate. The reaction was stopped by adding 50 μL of 4 M H2SO4 to each well, and the optical density was measured with a micro-ELISA plate reader (Coulter Microplate Reader UV Max) at a wavelength of 492 nm.
