Coating buffer

Cell-free primary RA synovial fluid and plasma from healthy blood donors were used for detecting IL6 (BD OptEIA), IL8 (BD OptEIA), IL-17 (peptrotech), TNFα (Pierce-endogen) and RANTES (Ray Biotech) by the BD OptEIA enzyme-linked immunosorbent assay (ELISA) Kit following the manufacturers’ protocol. Briefly, non-tissue culture treated 96-well plates (Becton Dickson) were coated with capture antibody diluted in coating buffer (eBioscience) and incubated overnight at 4°C. PBS (Mideatech, Inc.) 10% FBS (Atlanta Biologicals) was used for blocking and as assay diluent as specified in the protocol. All washes during the protocol were performed with a Bio-Rad wash machine (Model 1575 Immunowash) using PBS with 0.5% Tween-20 (Sigma). After adding substrate, a color reagent TMB substrate also from BD biosciences, samples were stopped with the TMB stop solution (Kirkegaard and Perry Labs). Plates were read in a Spectramax PLUS 384 spectrophotometer at 450 nm with a background subtraction wavelength of 570 nm.

Serum titers against ID97 antigen were evaluated by antibody-capture ELISA. Briefly, Corning high-binding 384-well plates (VWR International, Radnor, PA, USA) were coated overnight at 4 °C with 2 µg/mL ID97 in coating buffer (eBioscience, San Diego, CA, USA), then washed in phosphate-buffered saline (PBS)-Tween 20. Serially diluted serum samples were incubated for 1 h followed by either anti-mouse IgG (H + L)-HRP, IgG1-HRP, or IgG2c-HRP (SouthernBiotech, Birmingham, AL, USA); and 3,3′,5,5′-tetramethylbenzidine (TMB) substrate was applied as per the manufacturer’s instructions. Plates were analyzed at 450 nm using an ELx808 Absorbance Reader (Bio-Tek Instruments, Winooski, VT, USA), and endpoints were set as the minimum dilution at which values were lesser than or equal to an OD of 0.5.

Blood was collected via cardiac puncture and the serum was isolated, aliquoted, and stored at −80 °C until use. Serum from each vaccination group were pooled to allow direct comparison between the enzyme-linked immunosorbent assay (ELISA)s and later SMFA. Individual ELISAs compared each vaccine group in triplicate. High-binding ELISA plates (Costar cat # 07-200-35, Sigma) were coated with Pfs25 protein at 1 μg/ml in coating buffer (eBioscience, San Diego, CA, USA) overnight at 4 °C. The plates were then blocked with 1× assay diluent (AD) (eBioscience 00-4202-56) for 1 h, washed with PBST (0.05% Tween), and added serially diluted serum (in 1× AD) for 2 h followed by anti-mouse IgG (Fc) (Abcam, ab97265, Cambridge, MA, USA) at 1:5000 for 1 h. 1xTMB substrate (eBioscience cat # 00-4201-56) was added and the reaction was stopped with 1 M sulfuric acid, before measuring the colourimetric change at 450 nm. Antibody endpoint titers (depicted as ELISA units) were determined using an absorbance value 3 standard deviations above the PBS-immunized control mice. For the IgG subclass ELISAs all steps were performed as described above except serum samples were added at one constant dilution (1:500) and the secondary antibodies (1:500 dilution) added were specific to IgG1, IgG2a or IgG3 (Southern Biotech, cat # 5300-05B, Birmingham, AL, USA).

Corning Costar 96-well EIA/RIA plate (Fisher Scientific, Thermo Fisher, UK) were coated overnight at 4°C with 1 μg/ml of HpBARI, HpARI or oxidation-resistant IL-33, diluted in 1X Coating buffer (eBioscience) (50 μl/well). Plates were washed three times with ELISA wash buffer (TBS+0.05% Tween 20) and blocked with 150 μl/well ELISA block buffer (PBS 0.5% BSA) for 1 hr at room temperature. Mouse or human ST2-Fc or IL-33-TRAP-Fc, or human IgG were diluted in block buffer starting at 11.1 nM and 10-fold diluted. Constructs were added to the protein-coated plate (50 μl/well) and incubated for 2 hr at room temperature. After incubation, the plate was washed four times with ELISA wash buffer. Anti-human IgG HRP (Invitrogen) diluted 1:3000 in ELISA block buffer (50 μl/well) was incubated for 1 hr at room temperature. Plate was washed four times and 1X TMB substrate was used (50 μl/well), stopping the enzymatic reaction with 2N H₂SO₄ (50 μl/well). Absorbance was read at 450 nm.

Peripheral blood was collect and subsequent centrifugation at 10,621 x g for 5 min to isolate the serum. Serum titers against ID93 were then evaluated by antibody capture ELISA. Briefly, Corning high bind 384 well plates (VWR International) were coated overnight at 4 °C with 2 µg/ml ID93 in coating buffer (eBioscience). Then, plates were blocked with 1% BSA-PBS and serum samples serially diluted. Detection antibodies were anti-mouse IgG2c (Southern Biotech Cat# 1079-05) or IgG1 HRP (Southern Biotech Cat# 1070-05). Plates were analyzed at 450 nm (ELx808, Bio-Tek Instruments Inc.) and endpoints were set as the minimum dilution at which values were lesser or equal to an OD of 0.2.

Briefly, 96-well microtiter plates were coated with polyclonal anti-collagen (Abcam, Cambridge, UK), anti-filaggrin (Santa Cruz Biotechnology, Dallas, Texas, USA), or anti-fibronectin (Abcam) antibodies (1:100 dilution) in 100μl of coating buffer (eBioscience) and incubated overnight at 4°C. The plates were washed seven times with PBS-T and incubated with assay buffer (eBioscience) for 1 h at room temperature (RT). The plates were again washed seven times with PBS-T. Serum samples (1:10 diluted in PBS) were added to each well in triplicate, and the plates were incubated for 2 h at RT. The plates were washed seven times with PBS-T, and then the 12G1 mAb (1:100 diluted in assay buffer) was added, and the plates were incubated for 2 h at RT. After incubation, the plates were washed seven times in PBS-T, 50µl of HRP-conjugated anti-mouse IgG antibody (1:3000, Amersham Pharmacia Biotech, Amersham, Buckinghamshire, UK) was added, and the plates were incubated for a further 1 hour at 37 °C. Finally, the plates were washed nine times, and the bound antibodies were visualized by adding substrate solution (eBioscience). Reactions were stopped after 15 mins by adding 50µl of stop solution (eBioscience). The colorimetric reaction was measured at 450 nm on a VersaMax ELISA reader.