BD Pharmingen™ Biotin Rat Anti-Mouse IgE

ELISA: For detection of mouse IgE by sandwich ELISA, this antibody may be used as the detection antibody at ~ 2 µg/ml coupled with purified rat anti-mouse IgE antibody (clone R35-72) (Cat. No. 553413) as the capture antibody.  Purified mouse IgE (Cat.No. 557079, 553481, or 557080)  may be used as the ELISA standard. Alternatively, the BD OptEIA™ Mouse IgE ELISA Set (Cat. No. 555248) is offered as a convenient sandwich ELISA product that is easy-to-use and may be used for the quantitation of soluble mouse IgE.

Mouse IgE ELISA protocol

I.   Coat with capture antibody:

1.   Dilute the purified rat anti-mouse IgE capture antibody (clone R35-72) (Cat. No. 553413) to ~ 2 µg/ml in coating buffer. Add 100 µl per well to an enhanced protein-binding ELISA-grade plate (e.g., BD Falcon™ Cat. No. 353279). Investigators are encouraged to to determine the optimal antibody concentration for their use. Titrations between 1-8 µg/ml are suggested.

2.   Shake plate to ensure all wells are covered by the 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 described section I, step 4.

III. Apply standards and samples:

1.   Leave column 1 of the plate as blank wells (i.e., no antigen added at 100 µl per well consisting of blocking buffer only). Use columns 2 and 3 for duplicates of the standard at 100 µl per well. Dilute the purified mouse IgE standard (Cat. No. 557079, 553481 or 557080) in blocking buffer.  Dilutions should range in a series of 8 two-fold dilutions, in blocking buffer, starting at 0.5 µg/ml. Use the remaining columns to add samples of interest at various dilutions in blocking buffer at 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.

IV.  Incubation with detection antibody:

1.   Dilute the biotinylated rat anti-mouse IgE antibody (clone R35-118) (Cat. No. 553419) to ~ 2 µg/ml in blocking buffer. Add 100 µl per well. Investigators are encouraged to to determine the optimal antibody concentration for their use. Titrations between 1-8 µg/ml are suggested.

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.

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) as recommended for the product (e.g 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.

VI.  Add substrate and develop:

1.   Thaw substrate (ABTS) buffer within 20 minutes of use. Add 11 µl of 30% H2O2 (Sigma-Aldrich, 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 50 µl per well of SDS/DMF Solution (optional).

2.   Read the plate at 405 nm.

1. Since applications vary, each investigator should titrate the reagent to obtain optimal results.
2. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
3. Caution: Sodium azide yields highly toxic hydrazoic acid under acidic conditions. Dilute azide compounds in running water before discarding to avoid accumulation of potentially explosive deposits in plumbing.