Capture antibody

One day prior to the ELISA testing of TNF-α secretion, MaxiSorp 96F-well plates (Nunc) were coated with 2 μg/ml capture antibody (eBioscience Inc., San Diego, CA, USA) and stored at 4°C, overnight. All incubations were at room temperature with shaking, and plates were washed with Tris-buffered saline (TBS) (pH 7.4, 0.05% Tween-20) between each step. Two hundred μl blocking buffer was added to each well before a 1-hour incubation. TNF-α samples were diluted at 1 : 4 and 1 : 10; TNF-α was added to each well before 2 hours of incubation. Biotin coupled anti-human antibody (eBioscience Inc., San Diego, CA, USA) was diluted in TBS + 1% bovine serum albumin (BSA) to 3 μg/ml and added to each well and subsequently incubated for 1 hour. Diluted ExtrAvidin®-Alkaline Phosphatase (Sigma-Aldrich) was added to each well prior to 30 min incubation. Finally, 100 μl pNPP substrate (Sigma-Aldrich, 1 mg/ml in 1 M buffer, pH 9.8) was added to each well and incubated for 45 min before the plates were read at 405 nm.

CFS samples were stored at −80 °C until use for cytokine quantification. All assays were performed in duplicate, and the sensitivity of the TGFβ ELISA was 9.4 pg/mL.
Sandwich ELISAs were performed in 96-well, flat-bottomed microtiter plates (Nunc-Immuno Plate Maxisorp, Roskilde, Denmark). Microplates were coated for 18 h at 4 °C with the capture antibody (eBioscience, San Diego, CA), washed three times with PBS-Tween 20 (0.05%), blocked for 30 min at room temperature with 2% PBS-BSA, and washed three times. All samples were treated to activate latent TGFβ to its immuno-reactive form. Twenty-microliters of 1 N HCl were added to 100 μL of each sample; samples were incubated for 10 min at room temperature and then neutralized with 20 µL of 1 N NaOH. The plates were then incubated at room temperature for 2 h. After washing, plates were incubated with the detection antibody (eBioscience, San Diego, CA) for 2 h at room temperature. Bound antibodies were detected using streptavidin-phosphatase conjugate (1:3000; Zymed Laboratories, San Francisco, CA) and p-nitrophenyl phosphate (Sigma, St. Louis, MO) as a substrate. Optical density readings were performed at 405 nm, after 30 and 60 min of incubation.

Concentrations of cytokines (IL-6, TNF, IL-10, IL-13, IL-4, and TGF-β1) in plasma samples or homogenized brain were determined using ELISA. We extracted the protein from the brain tissue of EAE mice by homogenizing the samples, using a Qiagen Tissue Ruptor device, in RIPA buffer with protease inhibitor for 1 minute on ice. Samples were centrifuged at 10,000 x g for 10 min, supernatants were collected, and total protein was measured using a Bradford protein assay (Bio-Rad, Mississauga). Levels of cytokines were normalized to total protein concentration. ELISA was then performed according to manufacturer instructions. Briefly, plates were coated with 2.5 µg/ml capture antibody (eBioscience) diluted in borate buffer (pH 8.2). Plates were blocked with blocking buffer (2% BSA in PBS) before samples were added to the plates and incubated overnight at 4°C. Next, biotinylated secondary antibodies (eBioscience) were added to the plate, followed by the addition of streptavidin-horseradish peroxidase. 3,3′,5,5′-Tetramethylbenzidine or substrate solution (TMB) (eBioscience) was added to the plate and incubated for 10–15 minutes before the reaction was stopped using 2N H₂SO₄ and the plate measured using an Epoch microplate spectrophotometer (Biotek, Winooski, VT).