Hrp conjugated streptavidin

In order to investigate the ability of the novel generated tribodies, in comparison with the corresponding parental mAbs, to compete in the PD-L1/PD-1 or LAG-3/MHC II (HLA-DRA) binding, competitive ELISA assays were performed by testing the binding of each biotinylated chimeric protein (PD-L1/Fc or MHCII) to PD-1 or LAG-3/GST-His, respectively, in the absence or in the presence of the unlabelled competitive tribodies. To this aim, NuncTM flat-bottom 96-well plate were coated with PD-1 or LAG-3 proteins. Then, the coated plates were pre-incubated with the unlabelled anti-PD-L1, anti-PD-1 or anti-LAG-3 antibodies at saturating concentrations for 2 h at room temperature (5:1 for anti-PD-L1 or -PD-1 and 3:1 for LAG-3 M/M excess ratio), and then further treated with the biotinylated PD-L1 or MHCII chimeric proteins, which were added to the plate at the same concentrations of the competitive antibodies for 2 h at room temperature. For the detection of bound biotinylated proteins, HRP-conjugated Streptavidin (Biorad, Segrate, Milano Italy) was added to the plate for 30 min and then the absorbance measured as mentioned above.

Human chimeric SARS-CoV-2 (2019-nCoV) Spike-RBD/Fc, SARS-CoV-2 (2019-nCoV) Spike-RBD/His and ACE-2/His proteins were purchased from Sino Biological, Dusseldorfer Eschborn, Germany. Human chimeric SARS-CoV-2 Omicron-RBD/Fc and IgG1 Fc proteins were purchased from R & D Systems, Minneapolis, MN, USA. Human chimeric ACE-2/Fc and SARS-CoV-2 Omicron-RBD/His proteins (from GenScript, Piscataway, NJ, USA) were also used.
Sotrovimab (from GlaxoSmithKline and Vir Biotechnology), Casirivimab and Imdevimab (from Regeneron Pharmaceuticals) mAbs were used. D3 mAb was expressed and purified as previously described [22 (link)].
HRP-conjugated anti-His antibody (Proteintech, Deansgate, Manchester, UK), anti-human IgG (Fab’)2 goat polyclonal antibody (Abcam, Banzarate, MI, Italy), anti-human Fc antibody (Sigma, St. Louis, MO, USA), and HRP-conjugated streptavidin (Biorad, Segrate, MI, Italy) were used for the detection of primary mAbs.
Human peptides derived from RBD portion of human SARS-CoV-2 Spike-RBD protein were synthesized from GenScript (Piscataway, NJ, USA) with the following aa sequences:
Peptide 1 Sequence: RKSNLKPFER;
Peptide 2 Sequence: GVEGFNCYFP;
Peptide 3 Sequence: RFASVYAWNRK;
Peptide 4 Sequence: RVQPTESIVR.

The following recombinant proteins were used: Human PD-L1/Fc, human PD-1/Fc and human LAG-3/Fc protein (all from Bio-Thecne R&D Systems, Inc., Minneapolis, MN, USA); Human LAG-3/His-GST and Human HLA class II histocompatibility antigen, DRA (from Cusabio Technology LLC, Houston, TX, USA); and Staphylococcal enterotoxin B (SEB), a toxin produced by the bacterium Staphylococcus aureus and used as stimuli for the activation of lymphocytes (Sigma, S4881 St. Louis, MO, USA).
The following antibodies were used: HRP-conjugated antihuman IgG (Fab’)2 goat polyclonal antibody (Abcam, Milan, Italy); anti-His-HRP-conjugated antibody (Proteintech, Deansgate, Germany); HRP-conjugated Streptavidin (Biorad, Milan, Italy); antihuman p44/42 MAPK (T202/Y204), antihuman Cleaved Caspase-3; anti-phospho-Sapk/Jnk (T183/Y185) rabbit polyclonal antibodies (all from Cell Signaling, Danvers, MA, USA); antivinculin monoclonal antibody (all from Santa Cruz Biotechnology, Inc. Dallas, TX, USA); and HRP-conjugate anti-mouse IgG and antirabbit secondary antibodies (all from Sigma, USA). PD-L1_1 (anti-PD-L1), PD-1_1 (anti-PD-1), LAG-3_1 (anti-LAG-3). Human IgG control (unrelated) monoclonal antibodies were produced in our laboratory, as previously described [32 (link)].