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IL-33 ELISA

 

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Human IL-33 ELISA Quantitation Kit

Catalog number: 40-288-23146

542 Euro / kit

For the quantitative determination of Human IL-33 levels in serum or

other biological samples.

This kit is for research use only, and is not for use in

diagnostic procedures.

GENTAUR BVBA Human IL-33 ELISA Kit Manual

_________________________________________________________________________________

Introduction

Interleukin-1 (IL-1) family members, such as IL-1α/β and IL-18, play important roles in host defense,

immune regulation, neuronal injury and inflammation (1, 2). Recently, a new member of this family, IL-

33, has been reported. It mediates its biological effects via IL-1 receptor ST2 and activates NF-kB and

MAPK pathways (3). IL-33 is synthesized in response to cytokines such as IL-1β and plays an

important role in TH2-associated immunology. IL-33 expression drives production of TH2-associated

cytokines from in vitro polarized TH2 cells and induces the expression of IL-4, IL-5, and IL-13 in vivo.

Overexpression of IL-33 in mice leads to severe pathological changes in mucosal organs. IL-33 was

previously identified as NF-HEV, a nuclear factor preferentially expressed in high endothelial venules

(HEV) (4). Recent studies revealed that IL-33 is a heterochromatin-associated nuclear factor and

endothelial cells constitute a major source of IL-33 mRNA in chronically inflamed tissues from patients

with rheumatoid arthritis and Crohn's disease(5). These data indicate that IL-33 is a dual function

protein that may function as both a proinflammatory cytokine and an intracellular nuclear factor with

transcriptional regulatory properties. The human IL-33 is expressed with a prodomain (aa 1-111) that

is cleaved by caspase-1 to produce a 18kDa protein (112-270) (6).

References

1. Lee, J. K. et al. Differences in signaling pathways by IL-1beta and IL-18. Proc Natl Acad Sci U S A

101, 8815-20 (2004).

2. Allan, S. M., Tyrrell, P. J. & Rothwell, N. J. Interleukin-1 and neuronal injury. Nat Rev Immunol 5,

629-40 (2005).

3. Schmitz, J. et al. IL-33, an interleukin-1-like cytokine that signals via the IL-1 receptor-related

protein ST2 and induces T helper type 2-associated cytokines. Immunity 23, 479-90 (2005).

4. Baekkevold, E. S. et al. Molecular characterization of NF-HEV, a nuclear factor preferentially

expressed in human high endothelial venules. Am J Pathol 163, 69-79 (2003).

5. Carriere V, et al. IL-33, the IL-1-like cytokine ligand for ST2 receptor, is a chromatin-associated

nuclear factor in vivo. Proc Natl Acad Sci U S A. 104, 282-7 (2007).

6. Martinon, F. & Tschopp, J. Inflammatory caspases: linking an intracellular innate immune system

to autoinflammatory diseases. Cell 117, 561-74 (2004).

Kit Contents:

1. Coating Antibody (Catalog # 15-288-23146)

Affinity-purified Chicken anti-human IL-33

Concentration: 1.0 mg/ml

Amount: 0.2 ml

Storage: -20ºC

2. Calibrator (Catalog # 10-288-23146)

Recombinant human IL-33 Antigen

Concentration: 1 mg/ml

Amount: 10 ul

Working Range: 500 – 0.7 ng/ml

Storage: -20ºC

3. HRP Detection Antibody (Catalog # 25-288-223146)

Affinity-purified Chicken anti-human IL-33–HRP Conjugate

Concentration: 0.6 mg/ml

Amount: 30 ul

Storage: 4ºC

*Buffers, Substrates and Plates are not included.

Notes:

1. Range of Detection: 500 – 0.7 ng/ml

2. Shelf life: Affinity purified IgY antibody is stable for at least two years if stored in

non-defrosting -20oC (not -80oC) freezer. HRP-conjugated IgY can be stored at 4oC

for six months or longer.

3. Assay Condition:

The kit performance has been optimized for the stated protocol using the materials

listed and standard dilutions from 500-0.7 ng/ml of human IL-33. For alternative

assay conditions, the operator must determine appropriate dilutions of reagents.

ELISA assay reactivity is sensitive to any variation in operator, pipetting and

washing techniques, incubation time and temperature, composition of reagents, and

kit age. Adjustments may be required to position the standard curve and/or samples

in the desired detection range.

4. Country of Origin: United States of America

5. Assay Use: For in vitro laboratory use only. Not for any clinical, therapeutic, or

diagnostic use or consumption in human or animals.

Human IL-33 Quantitative ELISA Protocol

Materials

1. TMB Peroxidase Substrate System. KPL, Cat. #50-76-00.

2. Costar 96 well EIA/RIA high binding plate (Corning, Inc. Cat No. 3590)

3. Sodium Bicarbonate. MW 84.01, Sigma-Aldrich, Cat. # S6014-5kg.

4. Tris Hydroxvmethy Aminomethano Hydrochloride. MW 157.6, Fisher. Cat. # BP153-1.

5. Tris Crystallized free base. MW 121.14, Fisher. Cat. # BP 152-5.

6. NaCl, MW 58.44, Fisher. Cat. # BP 358-10.

7. Tween 20, Sigma. Cat. # P-1379.

8. BSA, Sigma. Cat. # A3059-100G.

9. Sulfuric Acid, 4.0N. Labchem Inc., Cat. # LC 25830-2.

Buffer Preparation

1. Coating Buffer: 0.05 M Carbonate-Bicarbonate, pH 9.6

2. Wash Solution: 0.05% Tween 20 in PBS, pH 7.4

3. Blocking Solution: 50 mM Tris, 0.14 M NaCl, 1% BSA, pH 8.0

Sample/Conjugate Diluents: 50 mM Tris, 0.14 M NaCl, 1% BSA, 0.05% Tween 20, pH

8.0

Step-by-Step Method (Perform all steps at room temperature)

1. Coat with Capture Antibody

1) Determine the number of single wells needed. Standards, samples, blanks

and/or controls should be analyzed in triplicates.

2) Dilute 0.4 ul capture antibody to 100 ul Coating Buffer for each well to be

coated. Transfer 100 μl to each well. (Example: for 96 wells, dilute 40 μl capture

antibody to 10 ml Coating Buffer)

3) Incubate coated plate for 60 minutes.

4) After incubation, aspirate the Capture Antibody solution from each well.

5) Wash each well with Wash Solution as follows:

a. Fill each well with Wash Solution

b. Remove Wash Solution by aspiration

c. Repeat for a total of 3 washes.

2. Blocking

1) Add 200 μl of Blocking Solution to each well.

2) Incubate 60 minutes.

3) After incubation, remove the Blocking Solution and wash each well three times

as in Step 1.5).

3. Standards and Samples

1) Dilute the standards in Sample Diluent according to the chart below:

2) Dilute the samples, based on the expected concentration of the analyte, to fall

within the concentration range of the standards.

3) Transfer 100 μl of standard or sample to assigned wells.

4) Incubate plate 60 minutes.

5) After incubation, remove samples and standards and wash each well 5 times as

in Step 1.5).

4. HRP Detection Antibody

1) Dilute the HRP conjugate in Diluent. Recommended starting dilution is

1:1500. (Note: Adjustments in dilution may be needed depending on substrate

used, incubation time, and age of kit).

2) Transfer 100 μl to each well.

3) Incubate 60 minutes.

4) After incubation, remove HRP Conjugate and wash each well 5 times as in Step

1.5).

5. Enzyme Substrate Reaction

1) Prepare the substrate solution according to the manufacturer’s

recommendation.

2) Transfer 100 μl of substrate solution to each well.

3) Incubate plate 10-30 minutes.

4) To stop the TMB reaction, apply 100 μl of 4.0N Sulfuric Acid to each well. If

using another substrate, use the stop solution recommended by manufacturer.

6. Plate Reading

Using a microtiter plate reader, read the plate at the wavelength that is appropriate for

the substrate used (450 nm for TMB).

Calculation of Results

1. Average the triplicate readings from each standard, control, and sample.

2. Subtract the zero reading from each averaged value above.

3. Create a standard curve by reducing the data using computer software capable of

generating a four parameter logistic (4-PL) curve-fit. Other curve fits may also be

used.

4. A standard curve should be generated for each set of samples. See example below:

Step ng/ml Calibrator Sample Diluent

1 500 2 ul 4000 ul

2 166.7 300 ul from step 1 600 ul

3 55.6 300 ul from step 2 600 ul

4 18.5 300 ul from step 3 600 ul

5 6.2 300 ul from step 4 600 ul

6 2.1 300 ul from step 5 600 ul

7 0.7 300 ul from step 6 600 ul

Standards (ng /ml)

Smallest standard value: 0.141

Largest standard value: 2.386

Standard Curve

y = ( (A - D)/(1 + (x/C)^B ) ) + D: A B C D R^2

Std (Standards: Concentration vs MeanValue) 0.013 1.235 45.677 2.381 1

Technical Hints

1. The Capture antibody should be diluted with coating buffer immediately prior to its addition to

the wells. Coated plates are stable overnight at 4ºC when covered.

2. Change pipette tips between each addition of standard, sample and reagents to avoid crosscontamination.

3. Standards and samples should be pipetted to the bottom of the wells and all other reagents

should be added to the side of the wells to avoid contamination.

4. Ensure that all buffers are not contaminated or expired. When troubleshooting ELISA results,

it is recommended to prepare all new buffers in new vessels.

5. Do not add Sodium Azide to any of the buffers.

6. Sample and Conjugate dilutions should be made shortly before use.

7. Wash buffer should be aspirated from wells, as pouring wash buffer from wells may cause

cross-contamination.

8. When preparing dilutions, wipe excess antibody/analyte from pipette tips to ensure accurate

dilutions.

9. Incubation time of the Enzyme Substrate will depend on the substrate used and the intensity of

the color change. The high standard should have an O.D. reading of between 1 and 3. The

low standard should have an O.D. reading above background.

10. The Stopping solution should be added to the wells in the same order as the Enzyme

Substrate.

Troubleshooting

The following are some common problems encountered with the use of ELISA kits, and some of the

causes of these problems.

1. Problem: Low absorbance

Incorrect dilutions or pipetting errors.

Improper incubation times

Improper mixing of the TMB substrate. Each component is mixed in equal parts.

Wrong filter on microtiter reader. Wavelength should be 450 nm for TMB, 490 nm for

OPD, or 405 nm for ABTS.

Kit materials or reagents are contaminated or expired.

Incorrect reagents used.

2. Problem: High Absorbance

Cross contamination from other samples or positive control.

Incorrect dilutions or pipetting errors.

Improper washing.

Wrong filter on microtiter reader.

Contaminated buffers or enzyme substrate.

Improper incubation times.

Kit materials or reagents are contaminated or expired.

3. Problem: Poor Duplicates

Poor mixing of specimens.

Incorrect dilutions or pipetting errors.

Technical error.

Inconsistency in following ELISA protocol.

Inefficient washing.

4. Problem: All wells are positive

Contaminated buffers or enzyme substrate.

Incorrect dilutions or pipetting errors.

Kit materials or reagents are contaminated or expired.

Inefficient washing.

5. Problem: All wells are negative

Procedure not followed correctly.

Contaminated buffers or enzyme substrate.

Contaminated conjugate.

Kit materials or reagents are contaminated or expired.

 

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Copyright © 2002 GENTAUR Molecular Products
Last modified: 05/29/09