Quantification of Anti-rTsNd Antibodies

The levels of the specific total IgG, IgG1 and IgG2a antibodies to rTsNd in serum of immunized mice were determined by ELISA with rTsNd as described previously [30 (link)]. Briefly, microtiter plates (Nunc) were coated with rTsNd (1 μg/ml) in coating buffer overnight at 4°C, and blocked with 200 μl of PBS-0.1% Tween 20 (PBST) containing 5% skimmed milk. Then, 100 μl of immune serum with serial dilutions were added to each well and incubated at 37°C for 1 h. HRP-conjugated goat anti-mouse IgG antibodies (1:5000; Southern Biotechnology, USA) were added and incubated at 37°C for 1 h. The reactions were detected by addition of the substrate o-phenylenediamine dihydrochloride (OPD; Sigma) plus H₂O₂ and stopped with 50 μl/well of 2 M H₂SO₄. Absorbance at 490 nm was measured with a microplate reader (TECAN, Austria). All samples were run in duplicate.