Phage display selections, direct phage ELISAs and competitive phage ELISAs were performed as described [50 ] with minor modifications. Briefly, phage particles from the libraries were cycled through rounds of binding selections with SEB coated directly on 96-well Maxisorp immunoplates (Fisher Scientific, Nepean, ON, Canada) or biotinylated SEB captured by immobilized neutravidin (Thermo Fisher Scientific, Rockford, IL, USA) as the capture target. After four or five rounds of selections, phage particles were produced from individual clones grown in a 96-well format, and the culture supernatants were used in phage ELISAs to detect specific binding clones. Clones that bound specifically to SEB were subjected to DNA sequencing to decode the sequences of the displayed Fabs. To assess competition between Abs for binding to SEB antigen, a competitive phage ELISA was used, whereby binding of Fab-phage to immobilized SEB was assessed in the presence of solution-phase Fab protein, as described [50 ].
Maxisorp immunoplate
Manufactured by Thermo Fisher Scientific
Sourced in Denmark, United States
MaxiSorp immunoplates are high-binding polystyrene microplates designed for use in enzyme-linked immunosorbent assay (ELISA) and other immunoassay applications. The plates feature a MaxiSorp surface, which provides a high-affinity surface for immobilization of proteins and other biomolecules. The plates are available in various well configurations to accommodate different assay requirements.
Automatically generated - may contain errors
Lab products found in correlation
Tmb one component substrate, by Fortis Life Sciences (3 mentions)
Bovine serum albumin (bsa), by Merck Group (3 mentions)
Microplate reader, by Agilent Technologies (3 mentions)
Multiskan sky, by Thermo Fisher Scientific (3 mentions)
Horseradish peroxidase labeled goat anti rabbit immunoglobulins, by Medac (3 mentions)
Bradford reagent, by Bio-Rad (3 mentions)
A9544, by Merck Group (3 mentions)
Np50 bsa, by Biosearch Technologies (2 mentions)
R3 34, by BD (2 mentions)
Superblock t20 pbs blocking buffer, by Thermo Fisher Scientific (2 mentions)
Carbonate bicarbonate buffer, by Merck Group (2 mentions)
R35 95, by BD (2 mentions)
Neutravidin, by Thermo Fisher Scientific (2 mentions)
Mtp 900, by Corona Electric (2 mentions)
Elisa kit, by Thermo Fisher Scientific (2 mentions)
Hrp substrate opd, by Fujifilm (2 mentions)
Mab20.1 antibody, by Thermo Fisher Scientific (2 mentions)
Hrp conjugated anti mouse antibody, by Jackson ImmunoResearch (2 mentions)
Hrp conjugated anti rabbit antibody, by Santa Cruz Biotechnology (2 mentions)
Tetramethylbenzidine substrate, by BD (2 mentions)
76 protocols using maxisorp immunoplate
1
Phage Display for Anti-SEB Antibodies
2
Quantitative LRG1 ELISA Protocol
3
Selecting Wnt Receptor-Binding Fabs
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The phage-displayed synthetic library F was used to select for Fabs that bound to Wnt receptors, as described (Persson et al., 2013 (link)). Briefly, Fc-tagged ECD protein (R and D Systems) was immobilized on Maxisorp immunoplates (ThermoFisher, catalog number 12-565-135) and used for positive binding selections with library phage pools that were first exposed to similarly immobilized Fc protein to deplete non-specific binders. After 4 rounds of binding selections, clonal phage were prepared and evaluated by phage ELISA (Birtalan et al., 2008 (link)). Clones that displayed at least 10-fold greater signal for binding to antigen compared with Fc were considered to be specific binders that were subjected to further characterization.
4
Isolation of PCDH1-specific monoclonal antibodies
5
ELISA for Mouse Ig Isotypes
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Maxisorp immunoplates (Thermo Fisher Scientific) were coated with NP50-BSA (Biosearch Technologies) or antibodies against mouse Ig isotypes, followed by blocking with 0.5% BSA in PBS containing 0.05% Tween-20. Serially diluted sera were added to the wells, followed by incubation for 5–6 h at room temperature. After washing with PBS containing 0.05% Tween-20, HRP–conjugated antibodies against mouse Ig isotypes (SouthernBiotech) were added to the wells, followed by overnight incubation at 4°C. The HRP activity was detected with tetramethylbenzidine substrate (Kirkegaard & Perry Laboratories), and absorbance at 450 nm was measured using an iMark microplate reader (Bio-Rad) after quenching the reaction with 1 N HCl.
6
Quantifying LRG1 Protein by ELISA
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The LRG1 concentrations were measured by sandwich enzyme-linked immunosorbent assay (ELISA). Briefly, MaxiSorp immunoplates (Thermo Fisher Scientific, Waltham, MA, USA; #10547781) were coated overnight at 4 °C with 20 μg/mL of 15C4 mouse anti-LRG1 mAb in 0.2M NaCO3/NaHCO3 buffer pH9.4 (Sigma-Aldrich, Milano, Italy; #C3041). The 25 μL of samples and serial dilutions of human recombinant LRG1 as standard were diluted to 50 μL with a wash buffer (0.1% v/v Tween-20/PBS) and left to absorb in the wells overnight at 4 °C. Detection was performed with anti-LRG1polyclonal antibody (1:500; Sigma-Aldrich; #HPA001888), Biotinylated anti-rabbit IgG (1:40,000; Sigma-Aldrich; #B8895), Horseradish Peroxidase (HRP)-conjugated Streptavidin (1:200; Thermo Fisher Scientific; #S911), and Substrate Reagent kit (R&D Systems, Minneapolis, MN, USA); #DY999). The absorbance was measured at 450 nm with a 540 nm wavelength correction on a VersaMax microplate reader and a four-parameter logistic curve-fit was used to fit the standard curve.
7
Blocking CTLA-4 Binding to CD80/CD86
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The ability of Boch4C2 to block the binding of bovine CTLA-4 to CD80/CD86 was assessed using an ELISA. MaxiSorp Immuno Plates (Thermo Fisher Scientific) were coated with 1 μg/ml CD80-Ig or CD86-Ig in PBS for 30 min at 37°C and then blocked with PBS containing 1% bovine serum albumin (Sigma-Aldrich) for 30 min at 37°C. Biotin-conjugated CTLA-4-Ig was preincubated with Boch4C2, bovine IgG (Sigma-Aldrich), 4C2-D9, or mouse IgG1 (Southern Biotech) at molar ratios (CTLA-4-Ig:antibody)=1:0, 1:0.1, 1:0.5, 1:1, 1:2, 1:5, and 1:10 for 30 min at 37°C. These reagents were added to the microplate and incubated for 30 min at 37°C. Binding of biotin-conjugated CTLA-4-Ig to CD80-Ig or CD86-Ig was detected using the NeutrAvidin Horseradish Peroxidase Conjugate (Thermo Fisher Scientific) for 30 min at 37°C. TMB One Component Substrate (Bethyl Laboratories, Montgomery, TX, USA) was then added to the microplate, and the reaction was stopped using 0.18 M H2SO4. Absorbance at 450 nm was measured using an MTP-900 microplate reader (Corona Electric, Hitachinaka, Japan). Three duplicate independent experiments were performed. The relative binding of CTLA-4-Ig to CD80-Ig or CD86-Ig was calculated from the absorbance of samples preincubated with each antibody, and the absorbance of the sample preincubated without antibody, which was defined as 100%.
8
SARS-CoV-2 S1 Protein Binding Assay
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MaxiSorp immunoplates (ThermoFisher SCIENTIFIC, MA, USA) were coated overnight at 4 °C with varying amounts (200, 50, 12.5, 3.13, 0.78, 0.2, 0.05, and 0 ng/mL) of SARS-CoV-2 S1 protein (S1 subunit, Cat. No. 40591-V08H; Sino Biological, Beijing, China) in 100 μL coating buffer per well. The immunoplates were blocked for 1 h with blocking buffer (Cat. No. DS98200; Invitrogen, CA, USA), and then hACE2, CR3022, F26G19, or S1-mAb in 100 μL blocking buffer was added to each well and incubated for 1 h. After washing with wash buffer (Cat. No. WB01; Invitrogen), the bound receptor and antibodies were incubated with horseradish peroxidase (HRP)-conjugated anti-mouse IgG (1:2000; Cat. No. 31437; Invitrogen), anti-human IgG (1:2000; Cat. No. 31413; Invitrogen), or anti-rabbit IgG (1:2000; Cat. No. 31463; Invitrogen) detection antibody, as appropriate, for 1 h. After extensive washing with wash buffer, stabilized chromogen (TMB solution, Cat. No. SB01; Invitrogen) was added. The immunoplates were allowed to react for 10 min, and the reaction was stopped by addition of 1 M HCl. The optical density (OD) value was measured at 450 nm using a microplate reader (BioTek, VT, USA).
9
Measuring M2e-specific Antibody Responses
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Blood was collected from the lateral tail vein before vaccination, 10 days after each vaccination and 15 days after challenge. Titers of total M2e-specific serum IgG and f88- specific serum IgG were determined by ELISA using 96-well Maxisorp immuno-plates (Thermo, Nunc, U.S.) coated overnight with human consensus M2e2-24 peptide (Table 1 ) or purified f88ctr phages. All peptides were HPLC-purified and used at 2 μg/ml in carbonate buffer (3.39 g/L Na2CO3; 5.71 g/L NaHCO3; pH 9.6) at 50 μl/well at 37°C. After coating, plates were washed twice with PBS + 0.1% Tween20 and blocked with 1% bovine serum albumin (Sigma-Aldrich, U.S.) in PBS. The specific anti-M2e antibody titers in mouse serum were determined by incubating 1/3 serial dilutions in coated plates, starting with 1/100 dilution, for 1 hour. The wells were then washed and incubated with sheep anti-mouse IgG serum conjugated with horseradish peroxidase (HRP) (GE Healthcare UK Ltd.), or HRP-conjugated goat anti-mouse IgG1 and IgG2a (Southern Biotech, U.S.) for 45 min at room temperature. Finally, plates were washed and incubated with tetramethylbenzidine substrate (Sigma-Aldrich, U.S.) for 5 min and the reaction was stopped by adding 50 μl of 1 M H2SO4.
10
CD70 Costimulation Enhances T Cell Activation
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The costimulatory capability of CD70 protein on primary human T cells activation upon T cell receptor engagement was evaluated using plate-bound anti-CD3 antibody (Ultra-LEAF(TM) purified anti-human CD3 [OKT3], Biolegend, # 317326) as the TCR ligand. Anti-CD3 antibody and various concentration of CD70 protein in sterile PBS were added into MaxiSorp Immuno plates (Thermo Scientific, #439454) and incubated at 4 °C overnight. Next day the plates were washed three times with fresh PBS. Human Peripheral Blood pan-T cells (StemCell, # 70024) were added into the precoated plates and incubated at 37 °C, 5% CO2 for 3 days. The activation of T cells was evaluated by measuring the IFNγ production with V-PLEX Human IFN-γ Kit (Meso Scale Diagnostics, #K151QOD-4) following the manufacturer’s protocol. The data represent three independent biological replicates (mean± standard error).
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