At 48 h after the last OVA challenge, mice were anesthetized with isoflurane, and blood samples were obtained from the abdominal aorta and centrifuged at 3000 rpm for 10 min. Total IgE level in serum was determined by ELISA using purified rat anti-mouse IgE and biotinylated rat anti-mouse IgE as capture and detection antibodies, respectively (both from BD Pharmingen, San Diego, CA, USA). Standards were prepared using purified mouse IgE κ isotype control (BD Pharmingen). Optical density was determined by measuring the absorbance at 450 nm.
Biotinylated rat anti mouse ige
Manufactured by BD
Sourced in United States
Biotinylated rat anti-mouse IgE is a laboratory reagent used for the detection and quantification of mouse immunoglobulin E (IgE) in various experimental and analytical applications.
Automatically generated - may contain errors
Lab products found in correlation
Rat anti mouse ige, by BD (3 mentions)
Biotinylated rat anti mouse igg1, by BD (1 mentions)
Protein assay, by Bio-Rad (1 mentions)
Streptavidin hrp, by Thermo Fisher Scientific (1 mentions)
Streptavidin hrp, by BD (1 mentions)
Microplate autoreader el311, by Agilent Technologies (1 mentions)
Assayzap, by Biosoft (1 mentions)
Streptavidin horseradish peroxidase, by GE Healthcare (1 mentions)
Tween 20, by Merck Group (1 mentions)
2 2 azinobis, by Roche (1 mentions)
Hydrogen peroxide, by Chem-Supply (1 mentions)
5 protocols using biotinylated rat anti mouse ige
1
Quantification of Serum IgE Levels
2
Quantification of Immunological Markers in BAL Fluid
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C5a was measured in BAL fluid by ELISA. Purified rat anti-mouse C5a (clone I52–1486) was used as capture antibody, purified recombinant mouse C5a as standard and biotinylated rat anti-mouse C5a (clone I52–278) as detection antibody (all from BD Biosciences). Cytokines (IL-4, IL-5, IL-13), myeloperoxidase (MPO) and elastase were measured by ELISA (R&D systems, Minneapolis, MN, USA). Plasma total IgE was determined using rat-anti-mouse IgE as a capture antibody, purified mouse IgE as a standard, and biotinylated rat-anti-mouse IgE as detection (all from BD Biosciences) as described [24 (link)]. Plasma HDM-specific IgG1 was determined using HDM as capture and biotinylated rat-anti-mouse IgG1 as detection (BD Biosciences). BAL fluid IgM was determined as described [24 (link)], using rat-anti-mouse IgM as capture antibody, purified mouse IgM as standard and biotinylated goat-anti-mouse IgM (all from BD Biosciences) as detection. Total protein in BAL fluid was measured using Bio-Rad protein assay (Bio-Rad Laboratories, Veenendaal, Netherlands).
3
Murine Model of Allergic Sensitization
4
Quantification of Allergen-Specific Antibodies
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Concentrations of IL-5 and IL-13 in lung extract were measured using a commercial enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems, Abingdon, UK). OVA-specific serum IgE, IgG1, and IgG2c levels were determined by ELISA. OVA (10 µg/ml) was coated onto 96-multiwell plates. Serum samples were diluted 1/10,000 for IgG1, 1/1,000 for IgG2c and 1/20 for IgE. Biotinylated rat anti-mouse IgE (BD Biosciences, San Jose, CA, USA) was applied, followed by Streptavidin-HRP (BD Biosciences) to quantify OVA-specific serum IgE. Peroxidase-conjugated rat anti-mouse IgG1 and IgG2c (Southern-Biotech, Birmingham, AL, USA) were used to quantify OVA-specific serum IgG1 and IgG2c respectively.
5
Quantifying Serum IgE Levels in Mice
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To assess immunoglobulin E (IgE) levels, the mice were bled via cardiac puncture 24 hours after the last HDM challenge. Serum was obtained by centrifugation of blood samples at 9300×g for 5 minutes. The supernatant was removed and stored at −80°C. The total IgE was measured by ELISA. Briefly, ELISA plates (Affymetrix eBioscience, San Diego, CA, US) were coated overnight with 2 µg/mL purified anti-mouse IgE (BD Biosciences, San Jose, CA, US). Plates were washed with 0.05% Tween 20 (Sigma-Aldrich, NSW, AU) in PBS between each step. Plates were blocked with 1% BSA in PBS before addition of purified mouse IgE isotype standard monoclonal antibody (BD Biosciences) or mouse serum. IgE was detected using biotinylated rat anti-mouse IgE (BD Biosciences) at concentration of 2 µg/mL. Streptavidin-horseradish peroxidase (GE Healthcare Life Sciences, NSW, AU) was added followed by 2,2′ –azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (Hoffmann-La Roche, Basel, Switzerland) supplemented with 0.1% hydrogen peroxide (Chem-Supply, Adelaide, AU) to produce a colour reaction. This reaction was stopped by adding 20% wt/vol SDS (Bio-Scientific, Sydney, AU). Plates were read spectrophotometri-cally at an absorbance of 405nm (Microplate AutoReader EL311; BioTek, Winooski, VT, US), and data were analysed using linear regression analysis in AssayZap (Biosoft, Cambridge, UK).
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