Microtiter plate

SARS-CoV-2 spike protein-binding IgG, IgM, and IgA titers were determined by ELISA using remnant sera obtained from baseline (D –1) and 1, 3, and 7 days after patients received CCP (D1, D3, and D7, respectively). Briefly, microtiter plates (Costar, Corning) were coated with 25 μL of 2 μg/mL purified spike protein (32 (link), 55 (link), 75 (link)) in phosphate-buffered saline (PBS) overnight at 4°C, washed with 1× PBS/0.1% Tween (PBS-T), blocked with 3% (v/v) milk (Bio-Rad)/PBS-T for 1 hour at room temperature (RT), washed, and incubated with heat-inactivated sera for 2 hours at RT. Plates were then washed, incubated with isotype-specific HRP-labeled goat anti-human IgG (Thermo Fisher Scientific 31410), IgM (MilliporeSigma A6907), or IgA (MilliporeSigma A0295) for 1 hour at RT. Following final washes, plates were incubated with ultra-TMB ELISA substrate (Thermo Fisher Scientific), and color development was stopped by addition of 0.5 M sulfuric acid (MilliporeSigma). Well absorbances at 450 nm (A₄₅₀) were determined using a Cytation 5 (BioTek). The endpoint titer was determined as the highest dilution to give a signal 3 times the background A₄₅₀ (wells with no sera).

The collection of mouse blood samples in each group (n = 5) was conducted at 0, 14, 28, and 42 days, and the obtained serum samples were stored at −20°C in order to further evaluate antibodies and measure cytokines. Indirect ELISA was performed to detect IgG isotypes and specific anti-TvAP33 antibodies as described in a previous study with a few modifications (Zhang et al., 2018c (link)). In brief, microtiter plates (Costar, New York, NY, United States) were coated with recombinant TvAP33 in carbonate buffer (2.5 μg/ml, 100 μl/well) with a PH value of 9.6 overnight at 4°C and blocked with 4% BSA for 2 h at 37°C. A mouse serum dilution (ratio = 1/10, PBS was used as the diluent) was added to the wells, and the plates were incubated at 37°C for 2 h. After the plates were washed three times with PBST, the plates were treated with the HRP-conjugated secondary antibodies goat anti-mouse IgG2a, IgG1, and IgG (SouthernBiotech, Birmingham, AL, United States). Finally, 100 μl of 3,3,5,5-tetramethylbenzidine was added into each well, and then 100 μl (2 M) sulfuric acid was added to terminate the reaction. The light absorption at 450 nm was measured by an automatic ELISA reader (MULTISKAN FC, Thermo Fisher Scientific, Waltham, MA, United States) and all tests were completed in triplicate.

Cells were pretreated with various concentrations of four isolates (1–4) for 1 h and stimulated with LPS for 24 h. The cultured media of HT-29 cells and RAW 264.7 cells were collected for measuring the levels of IL-8, TNF-α, and IL-6, respectively. Microtiter plates (Costar, Corning, NY, USA) were added with coating buffer (14.2 mM Na₂CO₃, 34.9 mM NaHCO₃, and 3.1 mM NaN₃; pH 9.6) and incubated overnight at 4 °C. The plates were washed with PBST containing 0.05% Tween-20 (three times) and blocked with PBST containing 1% bovine serum albumin (BSA) for 1 h at 37 °C. The plates were supplemented with 100 μL of sample or standard IL-8 and incubated for 2 h at RT. The plate was washed with PBST and treated with 100 μL of biotinylated goat antimouse IgG and incubated for 1 h at 37 °C. The Streptavidin–horseradish peroxidase (HRP) conjugate (Amersham, Buckinghamshire, UK) was added to the plate. After treatment with 100 μL of tetramethylbenzidine, the plate was incubated for 30 min at RT and color emerged. The reaction was terminated with 4 M H₂SO₄, and OD values at 450 nm were obtained using an enzyme-linked immunosorbent assay (ELISA) reader.

The levels of antibodies in mouse sera were determined by enzyme-linked immunosorbent assay (ELISA) as previously described [25 (link)]. In brief, the microtiter plates (Corning Costar, Cambridge, MA, USA) were coated overnight at 4 °C with 10 μg /ml STAg in 50 mM carbonate buffer pH 9.6 (100 μl per well). After three washes, the plates were blocked with 3 % Bovine Serum Albumin (BSA) for 2 h at 37 °C and subsequently incubated with the mouse sera diluted 1:10 in PBS for 1 h at 37 °C. HRP-conjugated goat anti-mouse IgA, IgM, IgE, IgG, IgG₁, or IgG_2a (SouthernBiotech, Birmingham, AL, USA) was used as the secondary antibody to detect bound antibodies. Finally, the immune complexes were developed by incubation with 3,3,5,5-tetramethylbenzidine (TMB) for 20 min. The reaction was stopped by adding 2 M H₂SO₄, and the absorbance was measured at 450 nm with an automated ELISA reader (Multiskan FC, Thermo scientific, Waltham, MA, USA). All samples were run in triplicate.