Hrp conjugated goat anti mouse igg

The levels of IgG, IgG1, and IgG2a in the sera of immunized mice were determined by indirect ELISA as described [22 (link)]. Briefly, each well of an ELISA plate was precoated with either purified UV-DENV-2 or NS1_1-279 (1 μg/well). Plates were washed and blocked with 1% BSA (w/v) in PBST before 100 µL of 2-fold serial diluted sera was added. After incubation for 2 h, DENV-2 or NS1_1-279-specific antibodies were detected using 100 μL of HRP-conjugated goat anti-mouse IgG (1:3000 dilution, Invitrogen) and the TMB substrate (Bio-Rad). The reaction was terminated, and the absorbance was read at 450 nm. The endpoint titer was calculated as described by Wang et al. [22 (link)].
The IgG1 and IgG2a subclasses were detected using HRP-conjugated goat anti-mouse IgG1 or IgG2a antibodies (1:4000, Southern Biotech, Birmingham, AL, USA) as probes. The levels of DENV-2- or NS1_1-279-specific IgG1 and IgG2a were reported as the OD value, as described by Wang et al. [22 (link)].

The tissue was homogenized in a weight-to-volume ratio of 1:3 in modified lysis buffer containing 200 mM sodium chloride, 20 mM Tris-HCl, pH 8.0, 1% NP-40, 1 mM EDTA, and protease inhibitors (Roche, Basel, Switzerland). The homogenate was centrifuged (16.000 rcf) to pellet the cell and tissue debris (maximally 15-25% of the total homogenate). The protein concentration in the supernatant (between 75 and 85% of the total homogenate) was measured with the RC-DC Protein Assay Kit (Bio-Rad, Hercules, CA). The concentration of anti-Nogo A antibodies in serum, CSF, and CNS tissue was measured with a sandwich enzyme-linked immunosorbent assay. Briefly, plates were coated with rabbit anti-mouse IgG (Invitrogen, Carlsbad, CA) as the primary antibody and blocked using 5% w/v skimmed milk powder in TBS-T buffer (Tris-buffered saline, 0.1% Tween 20) followed by incubation of samples. To detect the anti-Nogo A antibodies in samples, a HRP-conjugated goat anti-mouse IgG (Invitrogen) secondary antibody was used. The activity of horseradish peroxidase (HRP) was determined by a Pierce TMB ELISA Substrate kit (Thermo Scientific, Waltham, MA), and optical density of the HRP reaction was determined at 450 nm using a TECAN SPARK reader. The concentrations of anti-Nogo A antibodies in serum, CSF, and CNS tissue were then plotted against a standard curve of anti-Nogo A antibody.

Stx2B-specific IgG in plasma was analyzed by ELISA as previously described (Mejias et al., 2013 (link)). Briefly, 96-well MaxiSorp plates (Greiner Bio-One) were coated with 5 μg of purified Stx2B subunit conjugated to histidine per mL of 15 mM carbonate-25 mM bicarbonate buffer (pH 9.6) overnight at 4°C. Wells were washed with PBS-T and blocked with 2% bovine serum albumin (BSA) (Sigma) in PBS (blocking buffer) for 1.5 h at room temperature. After washing with PBS-T, the plates were incubated overnight at 4°C with 1/10 diluted mouse plasma. Wells were washed with PBS-T and the plates were incubated with 1/3000 HRP-conjugated goat anti-mouse IgG (Invitrogen) diluted in blocking buffer for 1.5 h at room temperature with orbital agitation. Then, the chromogenic substrate was added as described above. Results were expressed as OD₄₉₂, which represents the value obtained for each sample minus the OD₄₉₂ obtained for its non-specific binding. Plasma from not immunized nor infected BALB/c mice was used as negative control, while serum from BLS-Stx2 immunized BALB/c mice (Mejias et al., 2013 (link)) was used as positive control.

Samples were boiled for 5 min in protein dissociation buffer containing 5% (vol/vol) β-mercaptoethanol, 2% (wt/vol) sodium dodecyl sulfate (SDS), 10% (vol/vol) glycerol, 80 mM Tris-HCl (pH 6.8), and 0.01% (wt/vol) bromophenol blue. Proteins were resolved by SDS-polyacrylamide gel electrophoresis (PAGE) and were visualized by Coomassie brilliant blue staining.
For Western blot analyses, proteins were transferred from gels onto polyvinylidene difluoride (PVDF) membranes by standard blotting procedures. Membranes were saturated (overnight, 4 °C) with PBS–5% (wt/vol) nonfat dry milk and incubated (1 h, 37 °C) with either a rabbit hyperimmune serum against RHDV to detect VP60 protein, or monoclonal antibodies directed against the inserted foreign epitopes, as indicated. After several washes with PBS–0.05% Tween 20 (PBS-T), membranes were incubated (1 h, 37 °C) with HRP-conjugated goat anti-rabbit IgG (Pierce) or HRP-conjugated goat anti-mouse IgG (Invitrogen). Membranes were then washed extensively with PBS-T and developed with peroxidase substrate (0.05% 4-chloro-1-naphthol, 0.08% H₂O₂ [30%], and 20% methanol in PBS).