Protocols

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Mouse IgE ELISA Protocol

I. Coat with Capture Antibody:

1. Dilute the purified anti-mouse IgE capture mAb (cat. no. 553413, clone R35-72) to 2 µg/ml^a in coating buffer.^* Add 100 µl per well to an enhanced protein-binding ELISA plate (e.g., Falcon Labware plate, cat. no. 353279 or Nunc Immuno plate, cat. no. 12-565-136).
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 min 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 RT for 30 min.
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 (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^a standard (cat. no. 557079, clone C38-2; or cat. no. 553481, clone 27-74) or mouse IgE^a 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 RT or overnight at 4°C.^b
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^a in blocking buffer. Add 100 µl per well.
2. Cover the plate and incubate at RT for 1 hour.
3. Wash the plate 6X with PBS/Tween® , as in Section I, Step 4, of this protocol.

V. Add Streptavidin-Horseradish Peroxidase (SAv-HRP):

1. Dilute Avidin-HRP (cat. no. 554058) or Streptavidin-HRP (cat. no. 554066) 1:1000 in blocking buffer^c. Add 100 µl per well.
2. Cover the plate and incubate at RT for 30 min.
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 min 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 RT for 20-30 min. Color reaction can be stopped by adding 50 µl per well of SDS/DMF Solution* (optional).
2. Read the plate at 405 nm.

*SOLUTIONS:

Coating Buffer: PBS, pH 7.2 - 7.4

PBS/Tween ®: PBS pH 7.2-7.4, Tween ® -20, 0.05%

Substrate Buffer:
ABTS (3-ethylbenzthiazoline-6-sulfonic acid, Sigma Cat. No. A-1888), 150 mg
0.1 M citric acid (e.g., 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

SDS/DMF Solution:
40% SDS (80 g SDS in 200 ml dd H₂O)
Add 200 ml DMF (N.N-dimethyl formamide)

PBS Solution
NaCl, 80.0 g
Na₂HPO₄ , 11.6 g
KH₂PO₄ , 2.0 g
KCl, 2.0 g
Add H₂O to 10 liter
Adjust pH to 7.2 - 7.4

Blocking Buffer
PBS, pH to 7.2 - 7.4
10% Fetal calf serum or 1% BSA

NOTES:

For troubleshooting information and details on ELISA techniques, please visit the following website: http://www.bdbiosciences.com/pharmingen/OptEIA/downloads.shtml