3 3 5 5 tetramethylbenzidine (tmb)

Pooled sera (n = 5) were prepared from the whole-blood samples of each group. H4R-specific IgG levels in sera were measured via ELISA. Briefly, 96-well flat-bottomed ELISA plates (Greiner; Sigma-Aldrich, St. Louis, MO) were coated overnight at 4°C with H4R protein (5 μg/mL) diluted in PBS (pH = 7.2). The plates were incubated with blocking buffer (1% BSA in PBS) for 2 hr at 37°C. The pooled sera were diluted 1:50 in dilution buffer (0.5%, v/v, Tween-20 in blocking buffer), added to the plates, and incubated for 2 hr at 37°C. After rinsing with washing buffer, the plates were incubated with biotin-conjugated goat anti-rat IgG (diluted 1:1,000 in 1% BSA/PBS-Tween; Southern Biotechnology, Birmingham, AL) for 2 hr at 37°C. The plates were then washed and incubated with TMB (Amresco) for 30 min in the dark. A stop solution consisting of 2 M H₂SO₄ was added to stop the reaction, and the OD₄₅₀ was measured.

Mice (n = 10-14 per group) were anesthetized with isoflurane and bled from the orbital plexus just before sacrificed. Serum samples were stored at −80°C. Serum titers of four IgG subclasses against mTg were detected by ELISA as described in our previous studies [1 (link), 15 (link), 47 ]. Serially diluted (five-fold) serum samples were added into the 96-well ELISA plates (Nunc) which had been coated with mTg (1 μg/well) and blocked with PBS-1% BSA (Sigma), and then incubated at room temperature for 2h. Anti-Tg IgG subclasses were detected using horseradish peroxidase-conjugated goat anti-mouse IgG1 (1:30,000), IgG2a (1:30,000), IgG2b (1:15,000), or IgG3 (1:250) (all from Bethyl, TX, U.S.). Plates were developed with tetramethylbenzidine (TMB, Amresco, OH, U.S.) and absorbance at 450 nm was measured using a 96-well plate reader (Bio-Rad, CA, U.S.). These determinations were performed in duplicate and their mean values were used for statistical analysis.

As an antigen for the enzyme-linked immunosorbent assay (ELISA), the RBD protein produced in CHO-K1 cells and purified using affinity and ion-exchange chromatography methods (protein purity > 98%) was used [30 (link)]. RBD protein (1 µg/mL in 2 m urea) was adsorbed onto 96-well plates (Greiner Bio-One, Kremsmünster, Austria) at 4 °C overnight, then the plate was washed in PBS with 0.05% Tween 20 (PBST) and blocked with 1% casein solution in the same buffer for 60 min at room temperature. Then, sera were added to the wells in threefold serial dilutions, starting from 1:50, in blocking solution, and incubated for 60 min at room temperature. The plate was washed, and horseradish-peroxidase-conjugated rabbit anti-mouse IgG antibodies (Sigma, St. Louis, MO, USA) were added and incubated for 60 min at room temperature. The substrate 3,3’,5,5’-tetramethylbenzidine (TMB) (Amresco, Dallas, TX, USA) was introduced into the wells of the plate washed with PBST. Optical density was measured at a wavelength of 450 nm using a ChroMate-4300 microplate reader (Awareness Technology Inc., Palm City, FL, USA). Serum dilution was taken as the titer in ELISA, at which the optical density value was more than two times higher than that for the negative control (blocking buffer was added to the wells instead of serum).