Hrp conjugated goat anti mouse igg antibody

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)].

MaxiSorp flat-bottom 96-well plates (Nunc, Roskilde, Denmark) were coated with recombinant hexameric NS1 of DENV2 strain 16681 (1 µg/mL; Bio-Rad, Hercules, CA, USA) in 0.1 M carbonate/bicarbonate buffer (pH 9.6) overnight at 4 °C. To investigate the effects of NS1 conformation on antibody binding, additional plates were coated in the same manner with NS1 treated with 0.1% SDS (i.e., dimer) [45 (link)] or NS1 treated with 0.1% SDS and heated for 5 min at 95 °C (i.e., monomer) [15 (link)]. The next day, plates were washed thrice with PBS containing 0.05% Tween-20 (PBST) and blocked with 2% BSA/PBST for 1 h at 37 °C. Sera were heat-inactivated for 30 min at 56 °C, diluted 100-fold in 2% BSA/PBST, then added to the wells and incubated for 1 h at 37 °C. Plates were washed thrice with PBST and then incubated with HRP-conjugated goat anti-mouse IgG antibody (1:5000; Invitrogen, Waltham, MA, USA) for 1 h at 37 °C. Plates were washed thrice with PBST and developed with 3,3′,5,5′-tetramethylbenzidine (TMB; Sigma-Aldrich, St. Louis, MO, USA) for 15 min at RT. The reaction was stopped by the addition of 2 N H₂SO₄ and absorbance was read at 450 nm on a Spark microplate reader (Tecan, Männedorf, Switzerland).

Microtiter plates were pre-coated with rabbit SARS-CoV-2 RBD-polyclonal antibody (1:4000, Sino Biological, Beijing, China) overnight at 4 °C. The wells were washed with PBST, followed by incubation with 1% BSA (w/v) in PBST for 1 h at room temperature. After incubation, UV-inactivated SARS-CoV-2 (10⁴ plaque-forming unit (PFU)/well) was added to each well and incubated for 2 h. Wells were washed and then probed with diluted samples for 2 h. After washing, SARS-CoV-2-specific antibodies were detected by the addition of HRP-conjugated goat anti-mouse IgG antibody (1:3000, Invitrogen, Carlsbad, CA, USA).

CD8+ T cell depletion was performed as previously described [11 (link)]. p210 immunized mice implanted with a subcutaneous osmotic pump to infuse AngII as described above were intraperitoneally injected with 150μg of anti-CD8 antibody (53-6.7; eBioscience, San Diego, CA) or isotype rat IgG control (Sigma-Aldrich) 3 days before the first immunization and then twice a week thereafter until euthanasia. The efficiency of the depletion was assessed by flow cytometry. Immune response to the injected antibody was assessed using ELISA. Briefly, sera from 5 mice per group were pooled and diluted 1:400 based on prior optimization. Capture antigens used were either CD8 Ab or Isotype at a concentration of 20μg/ml. After blocking with 1% BSA for 2 hours, diluted sera were incubated with the capture antigens for 2 hours, washed, and incubated with HRP-conjugated goat anti mouse IgG antibody (1:5000, Thermo Scientific). The ELISA plates were washed and detection chromogen (ABTS, Southern Biotech) was applied until color development and read on a micro-plate reader at 405 nm.