IL-33 (Mouse Interleukin-33) ELISA Kit
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Assay range : 31.25-2000 pg/ml
Size: 96T/Kit
INTRODUCTION
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.Spiking:
Cell culture supernate samples were assayed by adding 90 µl of sample and 10 µl of spike stock solution calculated to yield the intended 0, 150, 750 or 1500 pg/ml spike concentration.
Sample
Spiked level
Expected (pg/ml)
Observed (pg/ml)
Recovery %
Pooled cell culture medium (4X)
Low spike (150 pg/ml)
96.4
111.4
115.6
Medium spike
(750 pg/ml)
696.4
719.4
103.3
High spike (1500 pg/ml)
1319.4
1343.4
101.8
E.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