BD, 557079, BD Pharmingen™ Purified Mouse IgE κ Isotype Control

Alternative Name: anti-TNP
Isotype: Mouse BALB/c IgE, κ
Immunogen: TNP-keyhole limpet hemocyanin
Application: ELISA, Isotype control (Routinely Tested)
Concentration: 0.5 mg/ml
RRID: AB_479637
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 C38-2 is useful as a standard in a mouse IgE sandwich ELISA. MOUSE IgE ELISA PROTOCOL Notes: 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 (e.g., 1 - 8 µg/ml). Incubation times are recommended conditions for optimal sensitivity. I. 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. *See Solutions section below. Add 100 µl per well to an enhanced protein-binding ELISA plate (eg, BD Falcon™ ELISA Plates, BD Labware Cat. no. 353279). 2. Tap 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. II. 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. III. Apply Standards and Samples : 1. Leave column 1 as blank wells (ie, 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. IV. 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. V. 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. VI. Add Substrate and Develop : 1. Thaw substrate (ABTS) buffer* within 20 minutes of use. Add 11 µl of 30% H2O2 (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.