BD, 553481, BD Pharmingen™ Purified Mouse IgE, κ Isotype Standard

Alternative Name: dansyl; DNS; 5-[dimethylamino] naphthalene-1-sulfonyl
Isotype: Mouse C.SW IgE, κ
Application: ELISA Standard (Routinely Tested)
Concentration: 0.5 mg/ml
RRID: AB_10050441
Storage Buffer: Aqueous buffered solution containing ≤0.09% sodium azide.
Regulatory Status: RUO
Preparation And Storage: Preparation And Storage The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography. Store undiluted at 4°C.
Recommended Assay Procedures: Recommended Assay Procedures Clone 27-74 is useful as a standard in ELISA. Please refer to the Mouse IgE ELISA Protocol below. MOUSE IgE ELISA PROTOCOL Coat with Capture Antibody: 1.    Dilute the purified anti-mouse IgE capture mAb (Cat. No. 553413, clone R35-72) to 2 µg/ml in coating buffer.* Add 100 µl per well to an enhanced protein-binding ELISA plate (e.g., BD Falcon™ ELISA Plates, BD Labware Cat. No. 353279). 2.    Shake plate to ensure all wells are covered by capture antibody solution. 3.    Cover the plate and incubate for 1 hour at 37°C or overnight at 4°C. 4.    Wash the plate 3X with PBS/Tween*. For each wash, wells are filled with 200 µl PBS/Tween and allowed to stand at least 1 minute prior to aspirating or dumping. As a final step, tap plate on paper towels to remove excess buffer. Blocking: 1.    Block the plate with 200 µl blocking buffer* per well. 2.    Cover the plate and incubate at room temperature for 30 minutes. 3.    Wash the plate 3X with PBS/Tween, as in Section I, Step 4, of this protocol. Apply Standards and Samples: 1.    Leave column 1 as blank wells (i.e., no antigen added, 100 µl per well blocking buffer only). Use columns 2 and 3 for duplicates of the standard, 100 µl per well: dilute purified mouse IgE standard (Cat. No. 557079, clone C38-2; or Cat. No. 553481, clone 27-74) or mouse IgE standard (Cat. No. 557080, clone C48-2) in a series of 8 two-fold dilutions, in blocking buffer, starting at 0.5 µg/ml. Use the remaining columns to add samples at various dilutions in blocking buffer, 100 µl per well. 2.    Cover the plate and incubate for at least 1 hour at room temperature or overnight at 4°C. 3.    Wash the plate 3X with PBS/Tween, as in Section I, Step 4, of this protocol. Incubation with Detection Antibody: 1.    Dilute biotinylated anti-mouse IgE (Cat. No. 553419, clone R35-118) to 2 µg/ml in blocking buffer. Add 100 µl per well. 2.    Cover the plate and incubate at room temperature for 1 hour. 3.    Wash the plate 6X with PBS/Tween, as in Section I, Step 4, of this protocol. Add Avidin- or Streptavidin-Horseradish Peroxidase (Av-HRP or SAv-HRP): 1.    Dilute Av-HRP (Cat. No. 554058) or SAv-HRP (Cat. No. 554066) 1:1000 in blocking buffer. Add 100 µl per well. 2.    Cover the plate and incubate at room temperature for 30 minutes. 3.    Wash the plate 6X with PBS/Tween, as in Section I, Step 4, of this protocol. Add Substrate and Develop: 1.    Thaw substrate (ABTS) buffer* within 20 minutes of use. Add 11 µl of 30% H 2O2 (Sigma, Cat. No. H1009) to 11 ml substrate buffer and vortex. Immediately add 100 µl per well and allow to develop at room temperature for 20 - 30 minutes. Color reaction can be stopped by adding 50µl per well of SDS/DMF Solution* (optional). 2.    Read the plate at 405 nm. *SOLUTIONS Coating Buffer PBS/Tween Substrate Buffer PBS, pH 7.2 - 7.4 PBS ABTS (3-ethylbenzthiazoline-6-sulfonic acid, Sigma Cat. No. A-1888) 150 mg Tween-20 0.05% 0.1 M citric acid (eg, Fisher anhydrous, Cat. No. A-940) 500 ml Adjust pH to 4.35 with NaOH pellets Aliquot at 11 ml per vial and store at -20°C PBS Solution Blocking Buffer NaCl 80.0 g PBS Na2HPO4 11.6 g Fetal calf serum 10% SDS/DMF Solution KH2PO4 2.0 g or BSA 1% 40% SDS (80 g SDS in 200 ml dd H2O) KCl 2.0 g Add 200 ml DMF (N.N-dimethyl formamide) ddH2O to 10 liter Adjust pH to 7.2 - 7.4 NOTES a.   In most cases, coating the plate with primary mAb at 2 µg/ml, 100 µl per well and detecting with the biotinylated secondary mAb at 2 µg/ml, 100 µl per well yields a very satisfactory signal. However, for optimal signal, researchers should titrate each mAb over a range of concentrations (eg, 1 - 8 µg/ml). b.   Recommended incubation conditions for optimal sensitivity. c.   Streptavidin/Avidin-HRP conjugate from another supplier may be substituted and diluted according to the manufacturer's recommendation.