Tetramethylbenzidine

For enzyme-linked immunosorbent assay (ELISA), 1 μg of purified glyceraldehyde 3-phosphate dehydrogenase (GAP-DH; Abcam, Cambridge, UK) or purified Ubiquitin (Boston Biochem, Cambridge, Massachusetts, USA) per well was immobilized on Maxisorp plates (Nunc) overnight at 4°C. Wells were blocked with PBS containing 0.1% Tween-20 and 1% NFDM for 2 h at RT. Next, 100 μl of 1:200 diluted serum was added overnight at 4°C. After 5 washes with PBS containing 0.5% NFDM, bound antibodies were detected with peroxidase-coupled rabbit-anti-human-IgG (diluted 1:5000) or goat-anti-human-IgA secondary antibody (1:5000) for 1 h at RT (both Sigma-Aldrich). The substrate tetramethylbenzidine (Sigma-Aldrich) was added for 20 min and absorbance at 620 nm was determined in an automated ELISA plate reader (LabSystems, Finland). For competitive inhibition, serum samples were pre-incubated with GPGRAPI-GST-fusion protein or synthetic GPGRAPI peptide (CGPGRAPIC; Life Technologies, Grand Island, NY, USA) for 20 min at RT. Wildtype-GST or a scrambled synthetic peptide (RPIGGAP; Life Technologies, USA) were used as negative controls, respectively.

At the end of the study, five mice were sacrificed and sera collected and tested for antibodies directed to bovine native CII by enzyme-linked immunosorbent assays (ELISAs). Plates were coated with CII (Chondrex) in carbonate buffer (pH 9.6) at 4 °C for 16 h. After blocking with 10% BSA (Sigma Aldrich, Milan, Italy) in PBS for 2 h, sera (100 µL) were added to the wells and incubated for 2 h at RT. After washing, biotin conjugated goat anti-mouse total IgG (Thermo Fisher Scientific, Milan, Italy) was added into the plate and incubated for 2 h at RT. After washing, streptavidin-HRP (Sigma Aldrich) was added for 30 min at RT. Then, tetramethylbenzidine (Sigma Aldrich, Milan, Italy) and H₂O₂ in citrate phosphate buffer were added to each well. The color reaction was stopped with 1N HCl. The absorbance was measured at 450 nm using a microplate reader.
The results are expressed as absorbance (optical density, O.D.) in experimental wells minus the background absorbance obtained in wells without CII.

The expression levels of TNF‐α, IL‐6 and TGF‐β were measured with ELISA using specific antibody (Santa Cruz Biotechnology, Dallas, TX, USA; Abcam, Cambridge, MA, USA). One hundred microliters of diluted antigen (rat serum, 2 μg·μL⁻¹ in phosphate‐buffered saline; PBS) was immobilized in a 96‐well microtiter plate (Nunc, Rockilde, Denmark) overnight at 4 °C. After incubation, the plates were washed three times in washing buffer (twice with PBS, once with Tris‐buffered saline and Polysorbate 20). One hundred microliters of blocking solution [5% skim milk (Sigma‐Aldrich) in PBS] was added to each well. Afterwards, 100 μL of primary antibody (1 : 1000; Santa Cruz Biotechnology, Abcam) in blocking solution was added, followed by incubation for 2 h at room temperature on a shaker. Plates were washed as before. One hundred microliters of secondary antibody‐tagged horseradish peroxidase (1 : 1000; Santa‐Cruz Biotechnology) in blocking solution was added to each well. The plates were incubated for 1 h at room temperature. Finally, the plates were washed four times and 50 μL of tetramethylbenzidine (Sigma‐Aldrich) was added. The color intensity of the solution was read in a microtiter reader at 650 nm.

24 hours after the final inhalation of OVA, blood samples were collected and tested by enzyme-linked immunosorbent assay (ELISA) using an ELISA kit (Abcam, UK) and based on the instruction of the manufacturer. 96 wells microtiter plates were coated with isotype-specific goat anti-mouse mAbs (IgE, IgG₁, IgG_2a) diluted in PBS and 2% bovine serum albumin (BSA) and incubated overnight at 4°C. Plates were extensively washed and serially diluted serum samples (100 μl/ well) were added in duplicate and incubated at 37°C for 2 hours in a humidified atmosphere. After washing with 0.1% Tween 20 in PBS (PBS-T), horseradish peroxidase-conjugated anti-mouse IgE (Pharmingen, Hamburg, Germany) (1:4,000), IgG₁ (1:5,000), or IgG_2a (1:5,000) was added to each well and incubated at room temperature for 1 hour. The plate was washed by PBS-T and developed with 100 μl tetramethyl benzidine (Sigma-Aldrich). Absorbance (450 nm) was measured with an ELISA plate reader (Molecular Devices Corporation, Sunnyvale, CA).

Sera were assayed for the presence of IgG antibodies to rh-α3NC1 by ELISA. Briefly, 96-well microtiter plates (Nunc MaxiSorp) were coated overnight with rh-α3NC1 (100 ng per well) in carbonate-bicarbonate buffer, pH 9.6. After blocking with 1% bovine serum albumin, the wells were incubated for 1 h with mouse sera diluted 1/5,000 for detection of total IgG, 1/2,000 for detection of IgG1, or 1/500 for detection of IgG2a and IgG2b. Secondary antibodies were alkaline phosphatase-conjugated goat anti-mouse IgG (Rockland Immunochemicals, Gilbertsville, PA, USA) and horseradish peroxidase-conjugated goat anti-mouse IgG1, IgG2a, IgG2b, or IgG2c (Bethyl, Montgomery, TX, USA). Plates were developed with p-nitrophenol phosphate or tetramethylbenzidine (Sigma, St. Louis, MO, USA) as substrate, and absorbance was read at 405 nm with a SpectraMax ELISA plate reader (Molecular Devices, Sunnyvale, CA, USA).

To investigate the presence of polyclonal B cell activation, sera from dogs with VL were tested by ELISA to detect the concentration of IgG antibodies reactive to two different antigens: KLH (Keyhole limpet hemocyanine) and OVA (Ovalbumin). Briefly, these antigens were diluted to 10 μg/mL in coating buffer (15 mM Na₂HCO₃, 28 mM NaHCO₃, pH 9.6), placed into wells on 96-well microtiter plates, incubated overnight at 4°C. Next, non-specific reactions were blocked with PBS, pH 7.2, containing 0.1% Tween 20 and 0.5% gelatin. The wells were then reincubated with canine serum at a dilution of 1:100 in PBS-gelatin. These dilutions had been shown to constitute the best serum concentrations to produce less false-negative and false-positive results. Wells were then washed with PBS, pH 7.2, containing 0.05% Tween 20. Peroxidase-conjugated rabbit anti-dog IgG (diluted 1:10,000) (Sigma-Aldrich, St. Louis, MO, United States) was added and the plates were incubated for one hour at 37°C. Reactions were developed with tetramethylbenzidine (Sigma-Aldrich, St. Louis, MO, United States) and H₂O₂ in 0.1 M acetate-citrate buffer, pH 5.0, then stopped with H₂SO₄ and read in a spectrophotometer with a 450 nm filter (Emax Precison Microplate Reader, Molecular Devices Corporation, Sunnyvale, CA, USA).

ELISA experiments with His-tagged SOSIP trimers were performed essentially as described before^{13 (link),51 (link)}. Purified proteins (1 µg/mL) were diluted in TBS and immobilized on 96-well Ni-NTA functionalized ELISA plates (Qiagen) by a 2 h incubation at room temperature. Following a double wash step with TBS to remove unbound trimers, serial dilutions of test antibodies in TBS/2% skimmed milk were added and incubated for 2 h. After 3 washes with TBS, HRP-labeled goat anti-human IgG (Jackson Immunoresearch) diluted 1:3000 in TBS/2% skimmed milk was added and incubated for 1 h, followed by 5 washes with TBS/0.05% Tween20. A developing solution (1% 3,3’,5,5’-tetramethylbenzidine (Sigma-Aldrich), 0.01% H₂O₂, 100 mM sodium acetate and 100 mM citric acid) allowed the colorimetric reaction, which was stopped by the addition of 0.8 M H₂SO₄. Finally, color development (absorption at 450 nm, OD₄₅₀) was measured to obtain the different binding curves.
Endpoint antibody titers from rabbit sera samples were determined using Ni-NTA His-Tag-capture ELISA similarly as above described^{6 (link),51 (link)}. However, in this case, sera was diluted in TBS/2% skimmed milk/20% sheep serum and the secondary antibody was an HRP-labeled goat antirabbit IgG (Jackson Immunoresearch).