IL-33 (Mouse Interleukin-33) ELISA Kit

IL-33 (Mouse Interleukin-33) ELISA Kit

SKU: 32750

 

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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

     

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