To identify the IgE‐binding ability of the purified allergens, Western blots were performed. Methods from Rao et al. (2016 ) and Mondoulet et al. (2005 ) were modified as follows. Peanut protein extracted from crude, fried, and boiled peanuts were analyzed by reducing SDS‐PAGE and then transferred to nitrocellulose membranes for 90 min at 80 V, then the membrane was placed in blocking buffer (5% (w/v) defatted milk powder dissolved in 100 mM phosphate buffer, pH 7.2) for 90 min. Pooled patient serum (diluted 1:10 (v:v) in blocking buffer) was used as IgE provider to incubate the membrane overnight at 4°C. After using TBST (50 mM Tris‐Cl, 150 mM NaCl, 1% Tween‐20, pH 7.5) to wash the membrane for five times, the membrane was incubated by goat anti‐human IgE‐HRP (catalog A9667, sigma) for 2 hours at 25°C (the goat anti‐human IgE‐HRP was diluted 1:5000 (v:v) in blocking buffer).The results were observed by enhanced chemiluminescense (ECL) detection system (Kangweishiji, Beijing, China).
Goat anti human ige hrp
Manufactured by Merck Group
Sourced in China, United States
Goat anti-human IgE-HRP is a secondary antibody conjugated with horseradish peroxidase (HRP). It is designed to detect and quantify human IgE in various immunoassay applications.
Automatically generated - may contain errors
2 protocols using goat anti human ige hrp
1
IgE-binding Profiles of Peanut Allergens
2
IgE Antibody Detection in Fish Allergy
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A 20 μg/mL solution of each B-cell synthetic peptide or negative control peptides were used to coat 96-well high-binding plates (50 µL/well) (Costar Corning Inc., New York, USA). Plates were incubated overnight at 4°C and then blocked with PBS/1% BSA (w/v) (200 µL/well) for 2 h at 37°C. After three washes with PBS/0.05% Tween 20, plates were incubated with 50 µL of the sera from different fish allergic patients and healthy donors (1:5 in PBS/1% BSA) for 1.5 h at 37°C. After three washes, bound IgE antibodies were detected using 50 µL per well of goat anti-human IgE-HRP (1:1000 in PBS/1% BSA) (Sigma-Aldrich, St. Louis, MO, USA) for 1.5 h at 37°C. After washing, the colorimetric reaction was developed following the addition of 50 µL of o-phenylene-diamine (Sigma), and stopped by the addition of 50 µL of 3 M sulfuric acid. The optical density (OD) was measured at 492 nm using an ELISA Microplate Reader (Multiskan TM GO, Thermo Fisher Scientific). Analyses were performed in triplicate. The OD value obtained for each B-cell synthetic peptide was corrected by subtracting the OD value determined in negative control peptide samples.
Finally, the mean intensities for each peptide epitope are reported.
Finally, the mean intensities for each peptide epitope are reported.
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