SARS-CoV-2 stabilized spike protein (BEI Resources, #NR-52397) was diluted 1 μg/mL in 0.05 M carbonate-bicarbonate buffer (pH 9.6) and adsorbed onto Nunc™ MaxiSorp™ ELISA Plates by incubating 100 ng/well overnight at 4°C. Antigen-coated plates were washed three times with wash PBST (0.01 M PBS + 0.05% Tween-20), and plates were blocked for 6 to 8 h at 4°C with PBSTBA (PBST + 1% BSA + 0.02% NaN3). Blocked plates were incubated overnight at 4°C with 102- to 106-fold diluted BAL fluid or sera from I.N. vaccinated mice and sera from I.M. vaccinated mice. Plates were washed three times with PBST. A secondary biotinylated anti-mouse IgA, IgG, IgG1, or IgG2a antibody (SouthernBiotech) was diluted 5000-fold in 5-fold diluted PBSTBA and was added to plates for 2 h at RT. Plates were washed three times with PBST. 5000-fold diluted streptavidin-conjugated horseradish peroxidase (strep-HRP, ThermoFisher) was incubated for 2 h at RT. Plates were washed six times with PBST. Ultra TMB-ELISA Substrate Solution (ThermoFisher) was added to wells and plates were incubated for 15 to 25 min at RT. 2 N sulfuric acid was added to each well, and absorbance was measured at 450 and 630 nm on a Synergy HT plate reader (BioTek) with Gen5 software.
Strep hrp
Manufactured by Thermo Fisher Scientific
Strep-HRP is a conjugate of Streptavidin (Strep) and Horseradish Peroxidase (HRP). It is designed for use in various bioanalytical and immunodiagnostic applications that involve the detection and quantification of biotinylated molecules.
Automatically generated - may contain errors
Lab products found in correlation
Synergy ht plate reader, by Agilent Technologies (2 mentions)
Ultra tmb elisa substrate solution, by Thermo Fisher Scientific (2 mentions)
Maxisorp elisa plate, by Thermo Fisher Scientific (1 mentions)
Prism 8, by GraphPad (1 mentions)
Nunc maxisorp, by Thermo Fisher Scientific (1 mentions)
Envision 2105 multimode plate reader, by PerkinElmer (1 mentions)
Tmb turbo, by Thermo Fisher Scientific (1 mentions)
Tmb substrate, by Thermo Fisher Scientific (1 mentions)
4 protocols using strep hrp
1
SARS-CoV-2 Spike Protein ELISA Assay
2
Quantitative ELISA for Mucin-TFF Interactions
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A solution of pMucin, pMucinred or pMucinred+GH89 (100 µl, 1 µg ml−1) in 100 mM bicarbonate buffer pH 9.4, was added to each well of a 96-well plate (flat-bottom Nunc MaxiSorp, ThermoFisher) and the plate gently nutated for 2 h at 25 °C. The wells were rinsed four times with 200 µl PBS-T (50 mM NaPi pH 7.4, 150 mM NaCl, 0.2% Tween-20) then blocked for 1 h at 25 °C with blocking buffer (50 mM NaPi pH 7.4, 150 mM NaCl, 5% w/v BSA). The wells were rinsed four times with 200 µl PBS-T then incubated with 100 µl blocking buffer containing TFF (various concentrations) and 1:1000 Strep-HRP (ThermoFisher) for 1 h at 25 °C. The wells were rinsed four times with 200 µl PBS-T then developed by adding 100 µl of TMB-Turbo (ThermoFisher) and incubated for 30 min at 25 °C. The reaction was quenched by the addition of 100 µl of 2 M H2SO4. Absorbance at 450 nm was read within 20 min using an EnVision 2105 Multimode Plate Reader (PerkinElmer). Data was analysed using Prism 8 (GraphPad).
3
Bcl-xL Binding Assay Using ELISA
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ELISA
plates were incubated
overnight at 4 °C with 1.5 nmol of anti-HA antibody per well.
Plates were washed with wash buffer [1× PBS + 0.1% (v/v) Tween-20]
and blocked with 1× PBS + 5% (w/v) BSA for 2 h. In each well,
100 μL of sample and 100 μL of 33 nM biotinylated Bcl-xL were added and incubated for 4 h. Plates were washed, incubated
with streptavidin horseradish peroxidase conjugate (Strep-HRP, Thermo
Scientific) for 1 h, washed, and incubated with TMB substrate (Thermo
Scientific). Reactions were stopped after approximately 10 min with
2 M sulfuric acid, and the absorbance at 450 nm was measured via a
plate reader (Molecular Devices).
plates were incubated
overnight at 4 °C with 1.5 nmol of anti-HA antibody per well.
Plates were washed with wash buffer [1× PBS + 0.1% (v/v) Tween-20]
and blocked with 1× PBS + 5% (w/v) BSA for 2 h. In each well,
100 μL of sample and 100 μL of 33 nM biotinylated Bcl-xL were added and incubated for 4 h. Plates were washed, incubated
with streptavidin horseradish peroxidase conjugate (Strep-HRP, Thermo
Scientific) for 1 h, washed, and incubated with TMB substrate (Thermo
Scientific). Reactions were stopped after approximately 10 min with
2 M sulfuric acid, and the absorbance at 450 nm was measured via a
plate reader (Molecular Devices).
4
Quantification of Spike-Neutralizing Antibodies
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Spike-neutralizing antibodies were quantified using a modified ELISA assay in a 384-well UltraCruz® ELISA high-binding plate (Santa Cruz Biotechnology). Diluted spike protein in 0.05 M carbonate-bicarbonate buffer (1 μg spike/mL, pH 9.6) was incubated in wells of a 384-well plate (50 ng/well) overnight at 4°C. Plates were washed three times with PBST, and plates were blocked in PBSTBA for 6–8 h at 4°C. Blocked plates were incubated overnight at 4°C with 102- to 106-fold diluted sera from I.M. vaccinated mice. Plates were washed three times with PBST. Plates were incubated for 2 h at RT with 500 ng/mL (25 ng/well) recombinant biotinylated human ACE-2 (R&D Systems, #BT933-020) diluted in PBSTBA. Plates were washed three times with PBST, and 5000-fold diluted strep-HRP (ThermoFisher) was incubated for 2 h at RT. Plates were washed six times with PBST, and incubated with Ultra TMB-ELISA Substrate Solution (ThermoFisher) for 20 min. The reaction was stopped with 2 N sulfuric acid, and absorbance was measured at 450 and 630 nm on a Synergy HT plate reader (BioTek) with Gen5 software. Absorbances were normalized by row to correct for 384-well plate effects; specifically, absorbances of samples at 450 nm were divided by absorbances of blank wells at 450 nm in each row.
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