1 step ultra tmb elisa solution

V1V2 protein scaffolds were directly coated onto high binding ELISA plates (Corning) overnight at 4 °C at 4 µg mL⁻¹. V2 peptides were C-terminally biotinylated and coated onto streptavidin ELISA plates. The plates with blocked with PBS (0.05% polysorbate 20, 5% milk powder) for 2 h, before being probed sequentially with five-fold serially diluted monoclonal antibodies (starting at 10 µg mL⁻¹⁾ and an anti-Fc HRP conjugate used a 1:10,000 dilution (Sigma, Cat. No. A0170-10ML) in PBS (0.05% polysorbate 20, 5% milk powder) for 1 h each. The plates were developed using 1-step ultra TMB-ELISA solution (ThermoFisher Scientific) followed by 1 M sulphuric acid and read at 450 nm.
Envelope pseudotyped viruses (PSV) were grown in HEK293T/17 cells by co-transfecting the C1080 envelope plasmid and a pSG3ΔEnv backbone. Cell culture supernatants were collected after two days and filtered through 0.45 µm. PSVs were purified through a 20% sucrose cushion at 40,000×g, resuspended in PBS, and coated directly onto high binding ELISA plates. Plates were washed and probed as above in PBS (10% FBS, 2–4% BSA).

hrRNASET2 binding to actin was quantified as modified from Liu et al. [22 (link)]. Human platelet actin (5 ug/ml) (Cytoskeleton, Denver, CO, USA) was diluted in carbonate-bicarbonate buffer (Sigma-Aldrich, St Louis, MO, USA), pH 9.5, and coated directly onto 96-wells COSTAR EIA/RIA plates (Corning, NY, USA) overnight at 4°C. The plates were then blocked with 5% (w/v) BSA in PBS containing 0.25% Tween-20 (PBST), at room temperature for 1 h, and subsequently incubated with hrRNASET2, at 1:2 serial dilutions in PBST, overnight at 4°C. Plates were washed three times with TBST, under continuous shaking for 10 min each, at room temperature and then incubated with rabbit anti-RNASET2 polyclonal affinity pure antibodies (GENEMED SYNTHESIS, San Antonio, TX, USA), diluted 1:500 in PBST at 37°C for 1 h. After three washing as described above, 0.8 μg/ml peroxidase-conjugated AffiniPure goat anti-rabbit IgG (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) in TBST was added and incubated under the same conditions. Plates were then washed twice with TBST and once with TBS, as described above. 1-Step Ultra TMB-ELISA solution (Thermo Scientific, Pierce Biotechnology, Rockford, IL, USA) was then added and optical absorbance was detected at 650 nm with an Infinite F50 multidetection microplate reader (Tecan, Grödig, Austria).

Total plasma anti-OVA IgG, IgG1, and IgG2c titers were determined by performing enzyme-linked immunosorbent assays (ELISAs) as described previously (17 (link)), with the following modifications. Briefly, 384-well plates were coated with 10 μg/mL OVA antigen solution (FUJIFILM Wako Pure Chemical Co., Ltd, Osaka, Japan) overnight at 4°C. The plates were washed and incubated for 1 h with blocking buffer (phosphate-buffered saline [PBS] containing 1% bovine serum albumin). After blocking, the plates were washed and incubated with 5-fold serially diluted plasma for 2 h. To detect the bound antibodies, the plates were washed and incubated for 1 h with horseradish peroxidase-conjugated anti-mouse total IgG, IgG1, or IgG2c Ab (Bethyl Laboratories, Inc., Montgomery, TX). After the plates were washed, 1-Step Ultra TMB-ELISA solution (Thermo Fisher) was added to each well to initiate the color reaction. The reaction was stopped after 5 min by the addition of 1 M sulfuric acid, and the optical density at 450 nm (OD₄₅₀) was measured using SpectraMax^® iD3 device (Molecular Devices, LLC, San Jose, CA). The titer was defined as the highest dilution factor with an OD value of > 0.1.

Surface expression of T toxins were quantitated by using ELISA as described (13 (link)). Oocytes injected with cRNAs of T-C6 variants bearing the c-Myc tag were blocked with bovine serum albumin and then bound with c-Myc–Tag monoclonal antibody conjugated with horseradish peroxidase (1 μg/mL) (Invitrogen). Oocytes were washed and then incubated with 50 μL of 1-Step Ultra TMB–ELISA solution (Thermo Fisher Scientific). The reaction was stopped by adding 50 μL of 2 M H₂SO₄. Surface ELISA signals were quantitated at 450 nm.