A 6.5 nM concentration of His-tagged versions of the BG505 or ConM SOSIP trimer immunogens in TBS was added to a 96-well Ni-NTA plate (Qiagen) for 2 h at RT. The plates were blocked for 30 min with TBS + 2% skimmed milk. Threefold serial dilutions of BG505-immunized rabbit sera, starting from a minimum of 1:200 dilution, were added in TBS + 2% skimmed milk + 20% sheep serum for a 2 h incubation at RT. For ConM-immunized rabbit sera a starting dilution of 1:500 was used. A 1:3000 dilution of HRP-labeled goat anti-rabbit IgG (111-035-144; Jackson Immunoresearch) in TBS + 2% skimmed milk was added for 1 h at RT. Up to this point, in between each step, the plates were washed three times with TBS. Next, after washing the plates five times with TBS + 0.05% Tween-20, developing solution (1% 3,3’,5,5’-tetranethylbenzidine (Sigma-Aldrich), 0.01% H2O2, 100 mM sodium acetate, and 100 mM citric acid) was added. Development of the colorimetric endpoint proceeded for 3 min before termination by adding 0.8 M H2SO4. The same procedure was used to measure binding Ab titers against the unmodified I53-50NP, except that the I53-50NP coating concentration was 3 nM and the serum starting dilution was a minimum of 1:1000.
Ni nta plate
Manufactured by Qiagen
The Ni-NTA plates are a laboratory equipment designed for the purification of histidine-tagged proteins. They contain a nickel-nitrilotriacetic acid (Ni-NTA) surface that binds to the histidine tags on the target proteins, allowing for their separation and concentration from complex mixtures.
Automatically generated - may contain errors
Lab products found in correlation
Goat anti human igg hrp, by Jackson ImmunoResearch (2 mentions)
Hrp labeled goat anti rabbit igg 111 035 144, by Jackson ImmunoResearch (1 mentions)
Hrp labeled goat anti rabbit igg, by Jackson ImmunoResearch (1 mentions)
3 3 5 5 tetranethylbenzidine, by Merck Group (1 mentions)
Spectramax id3, by Molecular Devices (1 mentions)
Hrp labeled goat anti human igg, by Jackson ImmunoResearch (1 mentions)
3 3 5 5 tetramethylbenzidine (tmb), by Merck Group (1 mentions)
1 step ultra tmb, by Thermo Fisher Scientific (1 mentions)
Hek293f cell, by Thermo Fisher Scientific (1 mentions)
Bright glo, by Promega (1 mentions)
Casein, by Thermo Fisher Scientific (1 mentions)
Hrp labelled goat anti human igg, by Jackson ImmunoResearch (1 mentions)
Robotic plate washer, by Agilent Technologies (1 mentions)
3 3 5 5 tetramethylbenzidine (tmb), by LGC (1 mentions)
Spectramax m5 plate reader, by Molecular Devices (1 mentions)
Hrp conjugated goat anti mouse secondary antibody, by Cell Signaling Technology (1 mentions)
13 protocols using ni nta plate
1
ELISA for Trimer Immunogen Binding
2
Measuring Antibody Binding to HIV-1 Trimer
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A 6.5 nM concentration of His-tagged 16055 SOSIP.v8.3 trimer in TBS was added to a 96-well Ni-NTA plate (Qiagen) for 2 h at RT. The plates were blocked for 30 min with TBS/2% skimmed milk. Three-fold serial dilutions of rabbit sera, starting from a 1:100 dilution (week 0,4,6,12,20) or 1:1000 dilution (week 22), were added in TBS/2% skimmed milk/20% sheep serum for a 2 h incubation at RT. A 1:3000 dilution of HRP-labeled goat anti-rabbit IgG (Jackson Immunoresearch) in TBS/2% skimmed milk was added for 1 h at RT. Between each step, the plates were washed three times with TBS. Finally, after washing the plates five times with TBS/0.05% Tween-20, developing solution (1% 3,3′,5,5′-tetranethylbenzidine (Sigma-Aldrich), 0.01% H2O2, 100 mM sodium acetate and 100 mM citric acid) was added. Development of the colorimetric endpoint proceeded for 1 min before termination by adding 0.8 M H2SO4. The same procedure was used to measure binding antibody titers against the unmodified I53-50NP, except that the I53-50NP coating concentration was 3 nM and the serum starting dilution was 1:500. Binding titers (ED50-values) were determined as the dilution of serum that gave 50% of the maximal response from a sigmoidal curve.
3
Quantifying α-Thrombin Binding to GPIbα
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The GPIbα stock
solution (250 μg/mL) was diluted with PBS buffer to various
concentrations. Each GPIbα solution (50 μL) was added
to a Ni-NTA plate (Qiagen, Venlo, Netherlands), followed by overnight
incubation at 4 °C. The plate was rinsed three times with 200
μL of PBS and incubated with 100 μL of 1% bovine serum
albumin for 30 min at room temperature (20–25 °C, RT).
After the removal of the blocking buffer, the plate was filled with
50 μL of the α-thrombin solution at various concentrations,
followed by incubation for 1 h at RT. Subsequently, the wells were
rinsed three times with the washing buffer. For labeling of the captured
α-thrombin, 50 μL of biotinylated primary antibody (Novus
Biologicals, Centennial, CO) was added to the wells, followed by incubation
for 1 h and five rinses. The wells were then treated with peroxidase-conjugated
streptavidin (Novus Biologicals), followed by a 30 min incubation
and five rinses. To generate the absorption spectra, 50 μL of
the chromogen substrate was added to each well, followed by incubation
for 10 min. The absorbance was measured at 570 nm at this point, and
the absorbance at 450 nm was measured after the addition of 50 μL
of the stop solution using a UV–vis microplate spectrophotometer
(SpectraMax iD3, Molecular Devices, LLC, San Jose, CA).
solution (250 μg/mL) was diluted with PBS buffer to various
concentrations. Each GPIbα solution (50 μL) was added
to a Ni-NTA plate (Qiagen, Venlo, Netherlands), followed by overnight
incubation at 4 °C. The plate was rinsed three times with 200
μL of PBS and incubated with 100 μL of 1% bovine serum
albumin for 30 min at room temperature (20–25 °C, RT).
After the removal of the blocking buffer, the plate was filled with
50 μL of the α-thrombin solution at various concentrations,
followed by incubation for 1 h at RT. Subsequently, the wells were
rinsed three times with the washing buffer. For labeling of the captured
α-thrombin, 50 μL of biotinylated primary antibody (Novus
Biologicals, Centennial, CO) was added to the wells, followed by incubation
for 1 h and five rinses. The wells were then treated with peroxidase-conjugated
streptavidin (Novus Biologicals), followed by a 30 min incubation
and five rinses. To generate the absorption spectra, 50 μL of
the chromogen substrate was added to each well, followed by incubation
for 10 min. The absorbance was measured at 570 nm at this point, and
the absorbance at 450 nm was measured after the addition of 50 μL
of the stop solution using a UV–vis microplate spectrophotometer
(SpectraMax iD3, Molecular Devices, LLC, San Jose, CA).
4
Release Kinetics of chABC-SH3 from XMC-Peptide
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The release of chABC-SH3 from XMC-peptide was measured as previously described [26] (link). Briefly, 100 μl of the XMC-peptide containing chABC-SH3 was injected into a 2 ml Eppendorf tube at 37 °C. After gelation, 400 μl of warm artificial cerebrospinal fluid (aCSF: 350 mM NaCl, 3 mM KCl, 0.8 mM MgCl 2 , 1.4 mM CaCl 2 , 1.5 mM Na 2 HPO 4 , 0.2 mM NaH 2 PO 4 , pH 7.4) was carefully added on top of the gel. At 0, 1, 2, 5, and 7 d the supernatant was completely removed and replaced with fresh aCSF. MC-peptide gels without chABC-SH3 were used as blanks for each time point. Release samples were analysed by ELISA using a 96-well Ni-NTA plate (Qiagen, Toronto, ON) and an anti-FLAG antibody coupled with HRP (1:5000) for detection. The absorbance levels were read on a TECAN plate reader at 405 nm with a wavelength correction at 650 nm. Protein concentration was calculated based on the linear range of a standard curve from the same plate.
5
ADP-Ribosylation Assay for His6-Tagged Proteins
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Prepare a 1 μM solution of the target protein in PBR.
Add 50 μL of the target protein solution to a Ni-NTA coated 96-well plate and incubate at RT for 1 h.
6
Quantifying Serum Antibody Responses in Immunized Mice
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ELISAs performed to analyze the serum antibody response in immunized mice were adapted from.36 (link) In short, hexahistidine (his)-tagged Envs (2 μg/mL) were captured on Ni-NTA plates (Qiagen) and left overnight at RT. The next day, after the plates were washed with TBS, the serum was diluted in 2% skim milk/TBS supplemented with 20% sheep serum (Biotrading) and incubated for 2 h at 37°C (starting dilution 1:100). Following three washes with TBS, a 1:3000 dilution of HRP-labeled goat anti-human IgG (Jackson ImmunoResearch) in casein was added for 1 h at RT. After washing the plates five times with TBS/0.05% Tween-20, develop solution containing 100 mM sodium acetate, 100 mM citric acid, 0.01% hydrogen peroxide, and 1% 3,3′,5,5′-tetramethylbenzidine (TMB, Sigma-Aldrich) was added. After a set amount of time, the colorimetric reaction was terminated by adding 0.8M sulfuric acid. In Figure 2 , values were normalized to the highest AUC obtained per time point in a particular assay.
7
SARS-CoV-2 Spike Protein ELISA
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SARS-CoV-2, SARS-CoV, MERS, HKU1, 229E and NL63 S His-tagged proteins were loaded at 2 μg ml−1 in TBS/2% skimmed milk (100 μl/well) on 96-well Ni-NTA plates (Qiagen) for 2 h at room temperature (RT). Three-fold serially diluted COVA NAb were then added onto the plates for 2 h at RT followed by the addition goat anti-human IgG-HRP (Jackson Immunoresearch) secondary Ab (1:3000) for 1 h at RT. The plates were developed for 3 min using TMB solution then stopped, optical densities measured at 450 nm on a spectrophotometer and data graphed using GraphPad Prism software (v8.3.0).
8
SOSIP Trimer ELISA Characterization
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For His-tagged SOSIP ELISA, SOSIP trimer creation was characterized previously in the respective references (version four (Zm197m v4.1, AMC008 v4.2, and B41 v4.2; [13 (link)]) and version five (BG505 v5.2; [14 (link)]). ELISA was performed using Ni-NTA plates from QIAGEN Inc. TRIS-buffered saline (TBS) buffer base, blocking with 2% milk, peroxidase-labeled secondary Ab, and 1-step Ultra-TMB (Thermo Fisher Scientific) substrate color development, as described previously [4 (link)]. Other direct protein ELISAs used Immulon 2-HB plates, sodium bicarbonate buffer, phosphate-buffered saline (PBS) buffer base, blocking with fetal bovine serum (FBS), peroxidase-labeled secondary Ab, and 1-step Ultra-TMB (Thermo Fisher Scientific) color development as described previously [4 (link)].
9
SARS-CoV-2 Spike Protein Binding Assay
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SARS-CoV-2, SARS-CoV, MERS, HKU1, 229E and NL63 S His-tagged proteins were loaded at 2 µg ml−1 in TBS/2% skimmed milk (100 µl/well) on 96-well Ni-NTA plates (Qiagen) for 2 h at room temperature (RT). Three-fold serially diluted COVA NAb were then added onto the plates for 2 h at RT followed by the addition goat anti-human IgG-HRP (Jackson Immunoresearch) secondary Ab (1:3000) for 1 h at RT. The plates were developed for 3 min using TMB solution and then stopped. Optical densities were measured at 450 nm on a spectrophotometer and data graphed using GraphPad Prism software (v8.3.0).
10
Production and Characterization of Monoclonal Antibodies
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MAbs were transfected into HEK293F cells (ThermoFisher) and purified by protein A (ThermoFisher) affinity chromatography as previously described.89 (link) ELISA characterization was performed by coating 2 μg/mL of Env onto 96-well Ni-NTA plates (Qiagen) and incubating overnight at RT as described above, with the exception that three-fold serial MAb dilutions were made in casein (starting at 1 μg/mL, 10 μg/mL or 100 μg/mL). Neutralization assays were performed as previously described.49 (link),90 (link) Briefly, neutralization experiments were set up to measure the ability of MAbs to reduce luciferase gene expression in adherent TZM-bl cells. These cells have been modified to include firefly luciferase genes, which are under control of an HIV-1 promotor. Virus was incubated with serially diluted MAbs for 1 h at 37 °C. 400 nM saquinavir and 40 μg/mL DEAE were added to the cells before addition of the MAb/virus mixture. Cells were grown for three days at 37 °C, after which cells were lysed and luciferase activity was measured using Bright-Glo (Promega) substrate on a GloMax Discover machine.
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