Tetramethyl benzidine

MaxiSorp 96-well plates (Nunc, Denmark, Roskilde) were coated with a test antigen solution (LMP1, MBP, OVA, recombinant LMP1 fragments, or recombinant MBP fragments; 5 µg/ml in 50 µl/well) in 100 mM carbonate buffer, pH 9.0 at 4°C overnight, and washed with three portions of a wash buffer (PBS with 0.1% Tween 20, pH 7.4, 300 µl/well). Unless otherwise specified, this washing was performed after each step. The wells were then blocked with 250 µl of dry milk in carbonate buffer and incubated at 37°C for 1 h. Serum samples were diluted in PBS containing 0.5% dry milk and 0.05% Tween 20. Respective monoclonal or polyclonal antibodies were used for each antigen as a positive control. The plates were incubated with primary antibodies at 37°C for 1 h. Then, 50 µl of HRP-conjugated anti-mouse anti-Fab antibodies (1:5,000) was added to each well, and the plates were incubated at 37°C for 1 h. After washing with five portions of the wash buffer, 50 µl of tetramethyl benzidine (Amresco, Solon, OH, USA) was added to each well, and the plates were placed in the dark for 5–15 min. The reaction was stopped by adding 10% phosphoric acid (50 µl/well). The OD₄₅₀ values were measured on a Varioskan Flash microplate reader (Thermo Scientific, Waltham, MA, USA).

Antibody titers to rPSOP25 were determined by ELISA on day 14, 35 and 52 after the first immunization as previously described [26 (link)]. Briefly, 96-well plates were coated overnight with purified rPSOP25 at 4 °C, and blocked with blocking buffer (0.05% Tween 20 in 0.1 M PBS, 1% bovine serum albumin, pH 7.4) for 2 h at 37 °C. The plates were then washed twice with PBS-T (0.05% Tween 20 in 0.1 M PBS, pH 7.4) and incubated with pooled mouse anti-rPSOP25 sera (1:200 dilution) in blocking buffer at 37 °C for 2 h. After two washes, the wells were incubated for 2 h at 37 °C with a 1:5000 dilution of HRP-conjugated goat anti-mouse IgG antibody (Invitrogen, Waltham, USA). After five final washes, tetramethyl benzidine (Amresco, Solon, USA) was added and the reaction was stopped by 2 mM H₂SO₄. The absorbance at 490 nm was measured with an ELISA plate reader.
For estimating the end point titer of immunized mice, sera from all mice in each immunization and control group were pooled and diluted from 1:200 to 1:204800 in a blocking buffer and incubated at 37 °C for 2 h. The end point titers of the total IgG corresponded to the highest dilution at which the OD490 value was higher than the cut-off value, which was defined as the mean of the pooled negative control antisera + 3 × standard deviation [29 (link)].