Half-area ELISA plates (Corning, Kennebunk, ME) were coated with 2.5 μg/mL Streptavidin in PBS for 2–3 h at RT, and blocked with 3% milk in PBS for 1 h at RT. After blocking, 1 μg/mL LIV LASV pfGP-TD was captured for 2 h at RT, followed by incubation with the indicated antibody concentration overnight at 4°C. The antibody GP solution was removed and plates were washed three times using PBS with 0.05% Tween 20, followed by treatment with PBS or 50 mM citrate/150 mM NaCl buffer adjusted to the indicated pH value for 1 h at RT. The plates were then washed three times with PBS-Tween20. Detection was performed using horseradish peroxidaseconjugated goat α-human IgG antibody reagent (KPL) for 1 h at RT. After an additional wash step, color was developed by the addition of 3,3,′5,5′ tetramethylbenzidine (TMB)-H2O2 (Thermo Fisher) for 5 min at RT. Development was stopped by the addition of 2N sulfuric acid. Color was read as absorbance (optical density) at 450nm.
3 3 5 5 tetramethylbenzidine tmb h2o2
Manufactured by Thermo Fisher Scientific
3,3,′5,5′ tetramethylbenzidine (TMB)-H2O2 is a colorimetric substrate used for the detection and quantification of peroxidase enzymes in various immunoassays and diagnostic applications. It undergoes an enzymatic reaction with peroxidase to produce a colored product that can be measured spectrophotometrically.
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Lab products found in correlation
2 protocols using 3 3 5 5 tetramethylbenzidine tmb h2o2
1
pH-dependent Antibody Binding Assay
2
pH-dependent Antibody Binding Assay
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Half-area ELISA plates (Corning, Kennebunk, ME) were coated with 2.5 μg/mL Streptavidin in PBS for 2–3 h at RT, and blocked with 3% milk in PBS for 1 h at RT. After blocking, 1 μg/mL LIV LASV pfGP-TD was captured for 2 h at RT, followed by incubation with the indicated antibody concentration overnight at 4°C. The antibody GP solution was removed and plates were washed three times using PBS with 0.05% Tween 20, followed by treatment with PBS or 50 mM citrate/150 mM NaCl buffer adjusted to the indicated pH value for 1 h at RT. The plates were then washed three times with PBS-Tween20. Detection was performed using horseradish peroxidaseconjugated goat α-human IgG antibody reagent (KPL) for 1 h at RT. After an additional wash step, color was developed by the addition of 3,3,′5,5′ tetramethylbenzidine (TMB)-H2O2 (Thermo Fisher) for 5 min at RT. Development was stopped by the addition of 2N sulfuric acid. Color was read as absorbance (optical density) at 450nm.
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