For the ELISA inhibition test, pooled sera from 5 Korean allergic rhinitis patients sensitized to HDM were used. First, ELISA plate wells were coated with 10 µg/mL of D. pteronyssinus or D. farinae whole-body extract (Hollister-Stier) in 50 µL of bicarbonate buffer (pH 9.6) overnight. Then, 50 µL of serum samples (1:4-diluted in 1% bovine serum albumin in phosphate buffered saline, pH 7.4), which were pre-incubated with various quantities of inhibitor (threefold serially diluted from 30 µg to 137.17 ng) were added to each well and incubated for 1 hour. Subsequently, IgE antibodies were detected using biotinylated goat anti-human IgE (Vector, Burlingame, CA, USA) and streptavidin-peroxidase (Sigma-Aldrich, St. Louis, MO, USA). The color was developed with 3,3',5,5'-tetramethylbenzidine (Kirkegaard and Perry Laboratories, Gaithersburg, MD, USA). The absorbance at 450 nm was measured after addition of 0.5 M H2SO4 to stop color development.
Biotinylated goat anti human ige
Manufactured by Vector Laboratories
Sourced in United States
Biotinylated goat anti-human IgE is a secondary antibody product. It is used to detect the presence of human immunoglobulin E (IgE) in various immunoassay applications.
Automatically generated - may contain errors
Lab products found in correlation
Streptavidin peroxidase, by Merck Group (4 mentions)
3 3 5 5 tetramethylbenzidine (tmb), by LGC (2 mentions)
Immunocap, by Thermo Fisher Scientific (1 mentions)
96 well microplate, by Corning (1 mentions)
Streptavidin peroxidase conjugate, by Merck Group (1 mentions)
Vmax microplate reader, by Molecular Devices (1 mentions)
Microplate, by Corning (1 mentions)
9 protocols using biotinylated goat anti human ige
1
ELISA Inhibition Test for HDM Allergy
2
Allergen-Specific IgE Quantification by ELISA
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Levels of allergen-specific serum IgEs were measured by ELISA. Each allergen (2 µg/mL for recombinant protein and 10 µg/mL for allergen extract) in 50 mM carbonate buffer (pH 9.6) was coated onto a microtiter plate and kept at 4℃ overnight. After blocking with 3% skim milk in phosphate-buffered saline containing 0.05% Tween 20 (PBST), plates were incubated for 1 hour with serum samples at 1:4 dilution. Subsequently, plates were incubated with biotinylated goat anti-human IgE at 1:1,000 dilution (Vector, Burlingame, CA, USA) for 1 hour and streptavidin-peroxidase (Sigma-Aldrich, Sydney, Australia) at 1:1,000 dilution for the detection of IgE antibodies. Plates were washed three times with PBST between each step. Color was developed using 3,3'5,5'-tetramethylbenzidine substrate solution (Kirkegaard Perry Laboratories, Gaithersburg, MD, USA), and then absorbance at 450 nm was measured. The mean plus 2 SD of absorbance values of sera from non-sensitized subjects was used as the cutoff value.
3
Serum Antibody Quantification for Jun a 1
4
Inhibition Immunoassay for Allergen IgE
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Allergen extract (10 μg/mL) was loaded to 96 well microplate (Corning Inc., NY, USA). After blocking with 3% skim milk, 1:4 diluted serum samples that were pre-incubated with various concentrations of inhibitors were incubated. IgE antibodies were detected by incubating with biotinylated goat anti-human IgE (1:1,000) (Vector, Burlingame, CA, USA), followed by streptavidin-peroxidase (1:1,000) (Sigma-Aldrich). The color was developed using 3,3',5,5'-tetramethyl-benzidine (Kirkegaard & Perry Laboratories, Gaithersburg, MD, USA) as a substrate. The enzyme reaction was stopped by the addition of 0.5 M H2SO4. The absorbance at 450 nm was determined. Percentage of inhibition was calculated by (1–absorbance with inhibitors/absorbance without inhibitor) × 100.
To investigate the role of Bet v 1-like allergens from various oak extracts, we performed inhibition ImmunoCAP. Serum samples were pre-incubated with various concentrations (0.016 to 50 μg/mL) of inhibitors. Subsequently, IgE to Bet v 1 (t215) was measured by ImmunoCAP (Phadia, Uppsala, Sweden) following the manufacturer’s instructions. The percentage of inhibition was calculated as described above (except that the IgE concentration was used instead of absorbance).
To investigate the role of Bet v 1-like allergens from various oak extracts, we performed inhibition ImmunoCAP. Serum samples were pre-incubated with various concentrations (0.016 to 50 μg/mL) of inhibitors. Subsequently, IgE to Bet v 1 (t215) was measured by ImmunoCAP (Phadia, Uppsala, Sweden) following the manufacturer’s instructions. The percentage of inhibition was calculated as described above (except that the IgE concentration was used instead of absorbance).
5
IgE Reactivity Assay for Recombinant Protein
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IgE reactivity toward the recombinant protein was detected by ELISA. Recombinant protein (2 µg/mL) was coated on a microplate overnight in 0.05 M carbonate buffer, pH 9.6. After blocking with 3% skim milk in PBS-containing 0.05% Tween 20 (PBST), serum samples (diluted 1:4 in PBST containing 1% bovine serum albumin) were added and the plate was incubated for 1 hr. IgE antibodies were detected by adding biotinylated goat anti-human IgE (1:1,000) (Vector, Burlingame, CA, USA) followed by a 1-hr incubation, and then streptavidin-peroxidase conjugate (1:1,000) (Sigma-Aldrich) was added and the plate was incubated for an additional 30 min. Color development was initiated by adding the substrate 3,3'5,5'-tetramethyl-benzidine (Kirkegaard & Perry Laboratories, Gaithersburg, MD, USA). Absorbance at 450 nm was measured after stopping the enzyme reaction by adding 0.5 M H2SO4. The mean absorbance plus 2 standard deviations of the sera from healthy controls was used as a cutoff value.
6
Quantifying Human IgE Levels via ELISA
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Enzyme-linked immunosorbent assay (ELISA) plates (Coster) were coated overnight with 50 μL/well of affinity purified goat anti-human IgE (Vector Laboratories) at 5 μg/mL in bicarbonate buffer. Plates were then blocked with 100 μL/well of 0.5% bovine serum albumin (BSA), incubated at 37°C for three hours and washed four times with phosphate buffered saline (PBS) + 0.05% Tween 20. Test sera (50 μL/well) diluted 1:1,000 were added to appropriate wells and incubated at 37°C for one hour. After incubation, 50 μL/well of biotinylated goat anti-human IgE (Vector Laboratories) diluted 1:10,000 with 0.5% BSA in bicarbonate buffer were added to the plates and incubated at 37°C for one hour. This was followed by the addition of 50 μL/well of alkaline phosphatase (ALP)-conjugated streptavidin (Mabtech) diluted 1:2,000 with 0.5% BSA in PBS + 0.05% Tween 20 and incubated at 37°C for one hour. Plates were finally developed with 50 μL/well of p-nitrophenyl phosphate (pNPP; Sigma), and the optical density values were read in a VMax Microplate Reader (Molecular Devices Corporation) at 405 nm. Total IgE concentrations were calculated from standard curves (human serum IgE from NIBSC and titrated threefold from 10 ng/mL over six duplicated wells) included on each plate.
7
IgE Cross-Reactivity in Hop Pollens
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Inhibition ELISA was performed to examine IgE cross-reactivity between Japanese and common hop pollen extracts as follows. Japanese hop pollen extract (10 µg/mL) was added to a microplate (Corning Inc., Corning, NY, USA). After blocking with 3% skim milk in PBS containing 0.05% Tween 20 (PBST), the plate was incubated overnight with 1:4-diluted pooled serum samples that were pre-incubated with various concentrations (0.00064–10 µg/mL) of pollen extract. Subsequently, IgE antibodies bound to Japanese hop were detected using biotinylated goat anti-human IgE (1:1000) (Vector, Burlingame, CA, USA) and peroxidase conjugated with streptavidin (1:1000) (Sigma-Aldrich, St. Louis, MO, USA). Color was developed using KPL SureBlueTM TMB Microwell Peroxidase Substrate (1-Component) (Kirkegaard & Perry Laboratories, Gaithersburg, MD, USA). The reaction was stopped by adding 0.5 M H2SO4, and absorbance at 450 nm was measured. Percent inhibition was calculated as (1-absorbance with inhibitors/absorbance without inhibitor)×100.
8
IgE Binding Inhibition by E58
9
Allergen Inhibition ELISA Assay
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The potency of the extracts was determined by inhibition ELISA. Each well was coated with 100 μL of 10 μg/ mL of each allergen extract in 50 mM sodium bicarbonate buffer (pH 9.6). Serum samples (1:4 dilutions; pooled sera from 10 patients, specific immunoglobulin E [IgE] to each allergen > 0.35 kUA/L), which were preincubated with various concentration of inhibitors (22 μL samples with 88 μL of sera) were added and incubated for 1 hour. IgE antibodies were detected with biotinylated goat anti-human IgE (Vector, Burlingame, CA, USA) and streptavidin-peroxidase (Sigma-Aldrich, Sydney, Australia). The percentage of inhibition was calculated as (1–Ai/A0) × 100, where Ai stands for the absorbance at 450 nm with inhibitor, and A0 for the absorbance without inhibitor.
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