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.

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

1. Cheong LY, Wang B, Wang Q, Jin L, Kwok KHM, Wu X, Shu L, Lin H, Chung SK, Cheng KKY, Hoo RLC, Xu A. Fibroblastic reticular cells in lymph node potentiate white adipose tissue beiging through neuro-immune crosstalk in male mice. Nat Commun. 2023 Mar 3;14(1):1213.