Cat No.: 32750 Other Names: DVS27-related Protein, IL1F11
Principle of the Assay
This assay is a quantitative sandwich ELISA. The microplate is pre-coated with a polyclonal antibody specific for mouse IL-33.
Standards and samples are pipetted into the wells and any mouse IL-33 present is bound by the immobilized antibody. After washing away any unbound substances, a biotin labelled polyclonal antibody specific for mouse IL-33 is added to the wells. After wash step to remove any unbound reagents, streptavidin-HRP conjugate (STP-HRP) is added. After the last wash step, an HRP substrate
solution is added and colour develops in proportion to the amount of mouse IL-33 bound initially. The assay is stopped and the
optical density of the wells determined using a microplate reader. Since the increases in absorbance are directly proportional to the
amount of captured mouse IL-33, the unknown sample concentration can be interpolated from a reference curve included in each
assay.
Assay Performance
A. Typical representation of standard curve
The following standard curve is provided for demonstration only. A standard curve should be generated for each set of sample
assay.
Mouse IL-33 (pg/mL) | Absorbance (450 nm) | Blanked Absorbance |
0 | 0.11 | 0 |
31.25 | 0.145 | 0.027 |
62.5 | 0.179 | 0.062 |
125 | 0.236 | 0.118 |
250 | 0.319 | 0.202 |
500 | 0.65 | 0.532 |
1000 | 1.2 | 1.083 |
2000 | 2.35 | 2.233 |
B. Sensitivity
The lowest level of mouse IL-33 that can be detected by this assay is 6.5 pg/mL.
C. Precision
Intra-Assay Precision (Precision within an assay)
Three samples of known concentration were tested 8 times on one plate.
Sample | Mean (pg/mL) | SD (pg/mL) | CV (%) |
1 | 1387.6 | 56.4 | 4.1 |
2 | 700.1 | 20.7 | 6.5 |
3 | 56.4 | 3.0 | 5.7 |
Intra-Assay Precision (Precision within an assay)
Three samples of known concentration were tested 8 times on separate plates.
Sample | Mean (pg/mL) | SD (pg/mL) | CV (%) |
1 | 1425.7 | 33.5 | 2.4 |
2 | 713.8 | 29.6 | 4.2 |
3 | 146.3 | 18.3 | 12.5 |
D. Linearity
To assess the linearity of the assay, samples containing and/or spiked with high concentrations of mouse IL-33 were serially diluted with the 1×Assay buffer to produce samples with values within the dynamic range of the assay.
Serial dilution | Measured (pg/mL) | Expected (pg/mL) | Recovery % |
Neat | 1427.7 | 1427.7 | 100 |
1:2 | 703.7 | 713.9 | 98.6 |
1:4 | 329.7 | 356.9 | 92.4 |
1:8 | 171.2 | 178.5 | 95.9 |
F. Validation
Cell culture supernates:
Lungs from mice were chopped into 1-2 mm pieces and cultured in 15 mL RPMI supplemented with 10% FBS, 50 μM β-ME, 100
U/mL penicillin, and 100 μg/mL streptomycin sulfate or stimulated with 1.0 μg/mL lipopolysaccharide (LPS) for 24 hours. Cell culture medium were removed and assayed for levels of mouse IL-33. In addition, we also collected the cell lysates and measured IL-33
levels and compared with the total protein levels in the cell lysates.
Sample (cell lysates) | IL-33 levels/ total protein (pg/mg) |
LPS-stimulated | 381.7 |
Unstimulated | 190.9 |