Horseradish peroxidase hrp conjugated streptavidin

PD-1/PD-L1 competitive ELISA (#72005, BPS Bioscience, San Diego, CA, USA) was performed as per the manufacturer’s protocol. As a positive control, an anti-PD-1 neutralizing antibody (#71120) was purchased from BPS Bioscience. Briefly, recombinant hPD-L1 protein (#71104, BPS Bioscience) was coated on the plates (0.32 cm², #3917, Corning, New York, NY, USA) at 1 μg/mL with phosphate-buffered saline (PBS, pH 7.4) and incubated overnight at 4°C. The plates were washed with PBS containing 0.05% Tween 20 (PBS-T) and blocked with PBS-T containing 2% (w/v) bovine serum albumin for 1 hour at room temperature (RT). The biotinylated hPD-1 (#71109, BPS Bioscience) of 0.5 μg/mL was added to each well and incubated for 2 hours at RT. The horseradish peroxidase (HRP)-conjugated streptavidin (#554066, BD Biosciences, San Jose, CA, USA) of 0.2 μg/mL was added to each well and incubated for 1 hour at RT. The relative chemiluminescence was measured using a SpectraMax L microplate reader (Molecular Devices, San Jose, CA, USA).

A rabbit polyclonal antibody (polyAb) against a synthetic peptide corresponding to H2-K^b (ab93364) (Abcam PLC., Cambridge, MA, USA) was used for immunoblotting. Mouse anti-H2-K^b/H2-D^b monoclonal antibody (mAb) (clone No. 28-8-6) (BioLegend, Inc., San Diego, CA, USA) was used for immunoblotting. Rat anti-mouse CD9 mAb (KMC8) and horseradish peroxidase (HRP)-conjugated streptavidin were purchased from BD Biosciences (San Jose, CA, USA). HRP-conjugated secondary antibodies (Sigma-Aldrich, St. Louis, MO, USA) were used for immunoblotting.

The method has been published^{11 (link),17 (link)–19 (link)}. In brief, recombinant human IFN-γ (BD Biosciences) was coated onto ELISA plates (Maxisorp, Nunc) at 4 °C overnight while the uncoated control wells had only PBS. On the day of the experiment, a pre-coated plate was washed, and heparinized plasma added at 1:100 dilution in duplicate, and incubated for 2 h at room temperature. After washing, biotinylated mouse anti-human IgG monoclonal antibody (clone G18–145: BD Biosciences) and horse radish peroxidase (HRP) conjugated streptavidin (BD Biosciences) was added to each well. After 1 h incubation, the color was developed by adding 3,3′,5,5′-Tetramethylbenzidine substrate (BD Biosciences). The results were calculated as the absorbance index by (O.D._test – O.D._uncoated)/O.D._uncoated.

Colons were flushed with cold PBS, opened along longitudinal axis, cut into 0.5 cm pieces and incubated for 24 h in RPMI 1640 supplemented with 10% FCS and antibiotics (n = 3–5 mice per group). Colon supernatants were collected and stored at −20°C. TNF-α serum levels were detected using a mouse TNF-α ELISA kit (Biolegend) according to the manufacturer's protocol. Mouse cytokines and chemokines were analyzed using Bio-plex Pro Mouse Cytokines 23-plex Assay (Bio-Rad) according to the manufacturer's protocol. IFN-γ levels in sera or colon supernatant were measured using an ELISA. Rat anti-mouse IFN-γ (Molecular Immunology, HZI) in coating buffer were incubated in 96 well plates (MaxiSorb TM Immunoplates, Nunc) overnight at 4°C. The 96 well plates were then blocked for 1 h, with 3% BSA in 0.05% Tween 20. Diluted sera or colon supernatant were distributed to the wells and incubated for 2 h at room temperature. IFN-γ was detected with biotinylated anti-mouse IFN-γ antibodies (Molecular Immunology, HZI). Biotinylated antibodies were bound with horseradish peroxidase (HRP) conjugated streptavidin (BD Pharmingen). Bound HRP was determined using o-Phenylendiamin (OPD) as substrate and the results were read using an ELISA-reader (BioRad 3550-UV microplate reader) at a wavelength of 490 nm.

The concentration of IFNγ in supernatant of co-cultured dendritic cells and T cells co-culture was analyzed using coating (clone A.N 18) and detection (clone R46A2) antibodies. In brief, coating rat anti-mouse IFNγ was incubated in 50 µL coating buffer in 96 well plates (MaxiSorb TM Immunoplates, Nunc) over night. The 96 well plates were then blocked for 1 h with 3% BSA in 0.05% Tween 20. Diluted sera were added to the wells and incubated for 2 h at room temperature. Biotinylated detection rat anti-mouse IFNγ was added and incubated for 1 h. Then horseradish peroxidase (HRP) conjugated streptavidin (BD) was added and incubated for 30 min and the bound HRP was detected with o-phenylenediamine (OPD) substrate in terms of absorbance at 490 nm using ELISA reader XFluor software (Tecan SUNRISE).