IL-5 and IL-13 were analyzed by ELISA as recommended by the manufacturer (Invitrogen). Total serum IgE was measured with the following reagents: anti-mouse IgE (BD pharmingen, capture antibody; 553413; 1/250), mouse IgE standard (SouthernBiotech; 0114-01), and Goat anti-mouse IgE-HRP (SouthernBiotech detection antibody; 1110-05; 1/16000). HDM and OVA-specific antibodies were analyzed by ELISA in plates that had been coated 3 h at 4 °C overnight with 100 μl HDM (50 μg/ml in PBS) or OVA (50 μg/ml in PBS), with nonspecific binding blocked by incubation for 1 h with 5% BSA. Wells were washed for three times and mouse serum samples diluted in assay diluent were added and incubated for 2 h. Wells were washed three times, then HRP Rat Anti-Mouse IgG1 (BD pharmingen; 559626; 1/250) or Goat Anti-Mouse IgG2c-HRP (SouthernBiotech; 1078-05; 1/4000) was added and incubated for 1 h. Wells were washed five times, and then were incubated with tetramethylbenzidine substrate until the reaction approached saturation. Linear regression analysis or measurement of absorbance was used to determine antibody titers or relative differences.
Goat anti mouse ige hrp
Manufactured by Southern Biotech
Goat Anti-Mouse IgE-HRP is a secondary antibody conjugated with horseradish peroxidase (HRP). It is designed for the detection and quantification of mouse immunoglobulin E (IgE) in various immunoassays.
Automatically generated - may contain errors
2 protocols using goat anti mouse ige hrp
1
Quantification of Allergy-Related Antibodies
2
Serum IgE and IgG1 ELISA for HDM Allergy
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Blood samples (100 µl per mouse) from the retro orbital venous plexus were collected using a heparinized capillary after the mice were anesthetized by isoflurane (RWD Life Science), and stored at room temperature for 2 h. The samples were centrifuged (3,000 × g, 10 min, 4°C) to isolate the serum and stored at −80°C until further analysis. The serum levels of HDM-specific IgE and IgG1 were assayed using an ELISA kit (cat. no. DY008; R&D Systems, Inc.) according to the manufacturer's protocol. The 96-well ELISA plates were coated overnight at 4°C with 100 µl of a 4 µg/ml solution of HDM in carbonate-bicarbonate buffer (pH 9.6; Sigma-Aldrich; Merck KGaA). ELISA was performed using the dilutions of each serum samples, and the dilutions used were 1/4 for sIgE and 1/500 for sIgG1. ELISA was prepared using the Reagent diluent (cat. no. DY995; R&D Systems, Inc.) and Goat Anti-Mouse IgG1-horseradish peroxidase (HRP; 1:2,000; cat. no. 1071-05; Southern Biotech) or Goat Anti-Mouse IgE-HRP (1:2,000; cat. no. 1110-05; Southern Biotech). The other steps were performed following the manufacturer's instructions.
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