Opd peroxidase substrate

The antibody response to antigen in the serum of immunized mice was determined by enzyme-linked immunosorbent assay (ELISA). Briefly, twofold serial dilutions of mouse sera (from 1:50 to 1:3200) were added to antigen-precoated plates (Costar Assay Plate Half Area, Corning) and incubated for 1 h at RT prior to incubation with an HRP-conjugated secondary antibody (anti-mouse IgG, IgG₁, IgG_2a or IgG_2b; Bethyl) for 1 h at RT. To develop the reaction, plates were washed and incubated with OPD peroxidase substrate (Sigma-Aldrich), and the reaction was stopped with 3 N HCl. The optical density was read in a Victor X4 microplate reader (PerkinElmer) at 490 nm. The concentration of unknown samples was determined by interpolation in accordance with a properly generated standard curve.

Humoral immune responses were assessed by indirect EnzymeLinked Immunosorbent Assay (ELISA). Total IgG, and antibody isotypes were measured from mouse serum. Briefly, 50 µg of L3 antigenic protein was dissolved in 50 mM carbonate buffer to coat Maxisorp (Millipore, Bedford, MA, USA) plates. After overnight incubation at 4 °C, plates were washed. To avoid unspecific interactions, wells were blocked with ChonBlock Buffer (Chondrex, Redmond, WA, USA) for two hours at room temperature. Serum samples were diluted at 1:50 in ChonBlock and incubated for two additional hours at room temperature. Plates were washed after incubation, and HRP-conjugated goat anti-murine IgG, or IgG1, or IgG2a antibodies (Jackson Immunoresearch, West Grove, PA, USA) were diluted in ChonBlock detection antibody dilution buffer (Chondrex, Redmond, WA, USA). Samples were incubated at room temperature for sixty minutes in secondary antibody diluted at 1:2,500. Chemiluminescence produced by the secondary antibody was measured at 450 nm using a plate reader after addition of OPD peroxidase substrate (Sigma Aldrich, St. Louis, MO, USA). The reaction was stopped using 50 µL of 3 M HCl.

The antibody isotypes IgG, IgG1, and IgG2a titers were measured from peripheral blood using an indirect Enzyme-Linked Immunosorbent Assay (ELISA). Serum was collected at days 0, 15, 30 and 45 post-immunization. Ninety-six-well polystyrene microtiter plates (Thermo Fisher Scientific Inc., MA) were coated with 1 μg/ml recombinant SH3-267 or ZM270 proteins in 0.05 M carbonate-bicarbonate buffer (pH 9.6) and left overnight at 4°C in a humid chamber. After washing the plates with PBS-0.05% Tween-20 (v/v) buffer, they were treated with 0.8% gelatin (w/v) in PBS-Tween-20 buffer for 1 h at 37°C in order to block non-specific sites. Serial two-fold dilutions of sera containing primary antibodies from test and control animals were added and incubated for 3 h at room temperature. Rabbit anti-mouse IgG, IgG1, and IgG2a secondary antibodies conjugated with horseradish peroxidase (US Biological, Life Sciences) at a dilution of 1:1000, were added and incubated for 45 min. The reaction was revealed using OPD peroxidase substrate (Sigma-Aldrich Co., MO) and stopped with 50 μl H₂SO₄ 2N. Results were read using a VictorX3 ELISA reader (PerkinElmer Chile LTDA, Santiago, Chile) at 490 nm. All assays were done in triplicate.

ELISA was performed to detect the binding of the SARS-CoV-2 RBD protein to the ACE2 receptor.
Microtiter plates were pre-coated with 10,000 µl of ACE2-hFc (CIM, Cuba) (5 μg/ml) in phosphate buffered saline (PBS) and incubated overnight at 4 °C. Plates were blocked with 200 µl/well of 4% of skim milk in PBS-Tween 0.05% (PBST) during 1 h, at 20–25 °C. Next, the blocking solution was discarded and 100 µl/well of samples was applied and incubated for 1 h at 20–25 °C. The bound protein was detected using 100 µl/well of RBD specific MAb CBSSRBD-S1 (CIGB, Cuba) (5 µg/ml in assay buffer) for 1 h at 20–25 °C. Next, 100µL/well of a peroxidase conjugated anti-mouse IgG antibody (Sigma, A2554) was added and incubated for 1 h at 20–25 °C. Then, the o-phenylendiamine dihydrochloride (OPD) peroxidase substrate (Sigma) was added and plates were light-protection incubated for 20 min at 20–25 °C. The reaction was stopped using 50 µl H₂SO₄ (Panreac, Spain). The optical density (OD) at 490 nm was measured using a ELISA microplate reader (Diareader, Germany). All incubations were followed by three washing steps with PBST.