Human IgG expression in NHP serum was quantified by ELISA. 96-well plates (NUNC) were coated with 50 µl/well anti-human H + L antibody cross-adsorbed to monkey at a concentration of 5 µg/mL and incubated overnight at 4°C. The following morning, plates were washed four times in PBS + Tween20 (PBST) wash buffer using plate washer (BioTek) and blotted on paper towels. 200 μL of casein blocking buffer was added to the wells and plates were incubated for 1 h at room temperature. Plates were washed as above. NHP sera were diluted 1:10, 1:30, or 1:90 in casein and 50 µl/well was added in duplicate. Purified mAbs COV2-2130-YTE and COV2-2196-YTE were both run individually as standards. Plates were incubated for 1 h at room temperature. Plates were washed again as above. Goat anti-human IgG cross-adsorbed to monkey conjugated to HRP (Southern Biotech) was diluted 1:8000 in casein and 50 µL added per well. Plates were incubated at room temperature for 30 min. Plates were washed again as above. Plates were developed using 100 µl/well TMB Turbo (ThermoFisher) and the reaction was stopped after 10 min with 100 µl/well 2N sulfuric acid. Plates were read at both 450 and 570 nm.
Tmb turbo
Manufactured by Thermo Fisher Scientific
TMB Turbo is a ready-to-use substrate solution for the detection of horseradish peroxidase (HRP) in enzyme-linked immunosorbent assays (ELISAs). It undergoes a color change reaction when in the presence of HRP, enabling the quantification of target analytes.
Automatically generated - may contain errors
Lab products found in correlation
Plate washer, by Agilent Technologies (3 mentions)
96 well plate, by Thermo Fisher Scientific (2 mentions)
Envision 2105 multimode plate reader, by PerkinElmer (2 mentions)
Nunc maxisorp, by Thermo Fisher Scientific (2 mentions)
Prism 8, by GraphPad (2 mentions)
Mouse anti monkey igg hrp, by Southern Biotech (1 mentions)
Strep hrp, by Thermo Fisher Scientific (1 mentions)
5 protocols using tmb turbo
1
Quantifying Human IgG in NHP Serum by ELISA
2
Quantifying Anti-COVID Antibodies in NHPs
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Monkey anti-drug antibodies against the human IgG were detected by ELISA. 96-well plates (NUNC) were coated with 50 μl/well COV2-2130-YTE and COV2-2196-YTE at a concentration of 5 μg/mL and incubated overnight at 4°C. The following morning, plates were washed four times in PBS + Tween20 (PBST) wash buffer using plate washer (BioTek) and blotted on paper towels. 200 μL of casein blocking buffer was added to the wells and plates were incubated for 1 h at room temperature. Plates were washed as above. NHP sera were diluted two-fold starting at 1:100 in casein and 50 μl/well was added in duplicate. Plates were incubated for 1 h at room temperature. Plates were washed again as above. Mouse anti-monkey IgG HRP (Southern Biotech) was diluted 1:8000 in casein and 50 μL added per well. Plates were incubated at room temperature for 30 min. Plates were washed again as above. Plates were developed using 100 μl/well TMB Turbo (ThermoFisher) and the reaction was stopped after 10 min with 100 µl/well 2N sulfuric acid. Plates were read at both 450 and 570 nm.
3
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).
4
SARS-CoV-2 RBD Binding Assay for DMAb Quantification
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Expression levels of individual DMAb clones was detected using recombinant SARS-CoV-2 RBD proteins K444A for quantification of DMAb-2196-YTE and DMAb-2196 and F486A for quantification of DMAb-2130-YTE and DMAb-2130. 96-well plates (NUNC) were coated with 50 µl/well each respective RBD at a concentration of 5 µg/mL and incubated overnight at 4°C. The following morning, plates were washed four times in PBS + Tween20 (PBST) wash buffer using plate washer (BioTek) and blotted on paper towels. 200 μL of casein blocking buffer was added to the wells and plates were incubated for 1 h at room temperature. Plates were washed as above. NHP sera were diluted 1:10, 1:30, or 1:90 in casein and 50 µl/well was added in duplicate. Purified mAbs COV2-2130-YTE and COV2-2196-YTE were both run individually as standards. Plates were incubated for 1 h at room temperature. Plates were washed again as above. Goat anti-human IgG cross-adsorbed to monkey conjugated to HRP (Southern Biotech) was diluted 1:8000 in casein and 50 µL added per well. Plates were incubated at room temperature for 30 min. Plates were washed again as above. Plates were developed using 100 µl/well TMB Turbo (ThermoFisher) and the reaction was stopped after 10 min with 100 µl/well 2N sulfuric acid. Plates were read at both 450 and 570 nm.
5
Quantification of Glycosidase Activity on Mucin Glycoproteins
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A solution of pMucin (100 µl, 5 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, Thermo Scientific) 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 50 µl blocking buffer containing dTFF1bio or dTFF3bio (16 nM) for 1 h at 25 °C. The wells were rinsed four times with 200 µl PBS-T then treated with 100 µl GH89 (0–512 nM) in blocking buffer for 20 h at 30 °C. The wells were rinsed six times with 200 µl PBS-T then probed with 100 µl Strep-HRP diluted 1:1000 in blocking buffer for 1 h at 25 °C. The wells were rinsed six 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) and an EC50 calculated using Prism 8 (GraphPad) and the mean from three independent experiments.
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