Human IL-13 Rα2 ELISA kit 

Catalog # LF-EK60037 (1 kit, 96 tests)
Sandwich Enzyme-Linked Immunosorbent Assay for Quantitative Detection of
human Interleukin 13 Receptor Alpha 2
For research use only
Not for diagnostic or therapeutic procedures

Contents
1. Introduction ······························································· 3
2. Principles of Method ····················································· 3
3. Intended Use ······························································· 4
4. Storage and Stability ····················································· 4
5. Chemical Hazard ························································· 4
6. Kit Contents ······························································· 5
7. Materials Required But Not Provided ······························· 6
8. Precautions ····························································· 6
9. Preparations ······························································ 7
1) Sample preparation ············································· 7
2) Preparation of reagents ········································ 7
10. Assay Procedure ·························································· 9
11. Characteristics ···························································· 10
1) Calculation of results ·················································· 10
2) Typical data ·························································· 10
3) Sensitivity ···························································· 11
4) Specificity ···························································· 11
12. Troubleshooting ··························································· 12
13. Reference ··································································· 13

1. Introduction

IL-13 receptor alpha 2, also known as CD213a2, is a subunit of IL-13 receptor complex. It is
encoded by the gene locus Xq13.1-q28 on chromosome X. An important characteristic of this
subunit is that it does not have a cytoplasmic domain. Even though, the IL-13 Ra2 binds to IL
13Ra2 with high affinity, the receptor appears not directly involved with a typical IL-13
stimulated signal transduction. Unlike IL-13aR1, the binding of IL-13Ra2 to IL-13 is IL-4R
independent and is not associated with Jaks and the phosphorylation and activation of Stats.
There are evidences that soluble IL-13Ra2 may serve as decoy receptor to dampen the cell
reaction to IL-13 stimulation.
IL-13Rα2 exists both on the cell membrane, and in soluble form. IL-13Rα2 is relatively rich
in hemopoietic or hypervascular tissues, while expressed in low level or not expressed in other
tissues. The gene was initially cloned from a kidney cell line and then found to be expressed in
brain, spleen, liver, thymus, and also in the bone marrow.
The research on IL-13Ra2 has led to the discovery of association of abnormal IL-13Ra2
expression with a number of pathological conditions. The IL-13Ra2 gene polymorphism was
found to be involved with susceptibility to Systemic Sclerosis (Brigitte Granel et al. 2006). The
overexpression of Il13ra2 gene has been found in brain tumors and ovarian cancer, making IL13Ra2 one of the candidates as biomarker and target for immunotherapy. In the research by Mitomu Kioi et al in 2006, IL-13 cytotoxin was highly cytotoxic to the IGROV-1 ovarian
cancer cell line in vitro, and it mediated significant antitumor activity against a xenografted
tumor model. The result suggested that IL-13Ra2 could be a promising target for ovarian cancer
therapy. Vaccine therapy of glioma was tested by vaccination using dendritic cells transfected
with Il13ra2 mRNA and this experiment induced strong anti-tumor effects in mice (Makoto
Saka et al. 2010).

2. Principles of Method

This assay applies the quantitative sandwich enzyme immunoassay technique. A monoclonal
antibody specific to IL-13 Rα2 has been pre-coated onto a microplate. Standards and samples
are then added to the appropriate plate wells with a biotin -conjugated antibody preparation
specific for IL-13 Rα2 and incubated. IL-13 Rα2 if present, will bind and become immobilized
by the antibody pre-coated on the wells and then be "sandwiched" by biotin conjugate. Avidin is
a tetramer containing four identical subunits that each has a high affinity-binding site for biotin.
After washing away any unbound substances, avidin-Horseradish Peroxidase (HRP) will be
added to each well and incubated. Following a wash to remove any unbound antibody-enzyme
reagent, a substrate solution is added to the wells and only those wells that contain IL-13 Rα2,
biotin conjugated antibody and enzyme-conjugated Avidin will exhibit a change in colour. The
colour development is stopped and the intensity of the colour is measured.

In order to measure the concentration of IL-13 Rα2 in the samples, this package of reagent
includes two calibration diluents (Calibrator Diluent I for serum/plasma testing and Calibrator
Diluent II for cell culture supernatant testing.) According to the testing system, the provided
standard is diluted (2-fold) with the appropriate Calibrator Diluent and assayed at the same time
as the samples. This allows the operator to produce a standard curve of Optical Density (O.D)
versus IL-13 Rα2 concentration. The concentration of IL-13 Rα2 in the samples is then
determined by comparing the O.D. of the samples to the standard curve.

3. Intended Use

The AbFrontier human Interleukin 13 Rα2 ELISA kit is to be used for the in vitro
quantitative determination of human Interleukin 13 Rα2 concentrations in culture supernatants.
This kit has been configured for laboratory research use only and is not to be
used in diagnostic or therapeutic procedures.

4. Storage and Stability

All kit components of this kit are stable at 2 to 8℃. Any unused reconstituted standard
should be discarded or frozen at -20 . Standard can ℃ be frozen and thawed one time only
without loss of immunoreactivity.

5. Chemical Hazard

- Stop solution: This reagent is an irritant to eyes, skin and mucous membranes. Avoid
contact with eyes, skin and clothing. Wear suitable protective clothing, gloves and eye
protection. In the event of contact with eyes or skin, wash immediately with plenty of
water.
- All reagents containing Sodium Azide also contain Thimerosal as a preservative.
Thimerosal contains Hg thus should be handled with great care.

6. Kit Contents

Contents Number Volume
IL-13 Rα2 Microtiter Plate 1 (in aluminum foil bag with desiccant)
Biotin Conjugate 1 6 mL
Avidin Conjugate 1 12 mL
IL-13 Rα2 STANDARD 2 vials (lyophilized)
CALIBRATOR DILUENT I 1 25 ml
CALIBRATOR DILUENT II 1 25 ml
WASH BUFFER (20X) 2 30 ml
SUBSTRATE A (H2O2) 1 10 ml
SUBSTRATE B (TMB) 1 10 ml
STOP SOLUTION 1 14 ml
Protocol booklet 1
Plate sealers 2
① IL-13 Rα2 Microtiter Plate
: Pre-coated with anti-human IL-13 Rα2 monoclonal antibody.
② Biotin Conjugate
: Anti-IL-13 Rα2 polyclonal antibody conjugated to Biotin.
③ Avidin Conjugate
: Avidin conjugate to horseradish peroxidase
④ IL-13 Rα2 STANDARD
: Recombinant human IL-13 Rα2 (12.8 ng/vial) in a buffered protein base with
Preservative, lyophilized.
⑤ CALIBRATOR DILUENT I
: Animal serum with buffer and preservative, for serum/plasma testing.
⑥ CALIBRATOR DILUENT II
: Cell culture medium with calf serum and preservative, for cell culture supernatant
testing.
⑦ WASH BUFFER (20X)
: 20-fold concentrated solution of buffered surfactant.
⑧ SUBSTRATE A
: Buffered solution with H2O2.
⑨ SUBSTRATE B
: Buffered solution with TMB.
⑩ STOP SOLUTION
: 2N Sulphuric Acid (H2SO4). Caution: Caustic Material!
Do not mix or interchange different reagents from various kit lots.

7. Materials Required But Not Provided
① Single or multi-channel precision pipettes with disposable tips: 10-100 ul and 50-200 ul
for running the assay.
② Pipettes: 1 ml, 5 ml 10 ml, and 25 ml for reagent preparation.
③ Multi-channel pipette reservoir or equivalent reagent container.
④ Test tubes and racks.
⑤ Polypropylene tubes or containers (25 ml).
⑥ Erlenmeyer flasks: 100 ml, 400 ml, 1 L and 2 L.
⑦ Microtiter plate reader (450 nm ± 2nm).
⑧ Automatic microtiter plate washer or squirt bottle.
⑨ Sodium hypochlorite solution, 5.25% (household liquid bleach).
⑩ Deionized or distilled water.
⑪ Disposable gloves.
⑫ Absorbent paper towels
8. Precautions
1) Do not substitute reagents from one kit lot to another. Standard, conjugate and
microtiter plates are matched for optimal performance. Use only the reagents supplied
by manufacturer.
2) Allow kit reagents and materials to reach room temperature (20-25°C) before use. Do
not use water baths to thaw samples or reagents.
3) Do not use kit components beyond their expiration date.
4) Use only deionized or distilled water to dilute reagents.
5) Do not remove microtiter plate from the storage bag until needed. Unused strips should
be stored at 2-8°C in their pouch with the desiccant provided.
6) Use fresh disposable pipette tips for each transfer to avoid contamination.
7) Do not mix acid and sodium hypochlorite solutions.
8) Human serum and plasma should be handled as potentially hazardous and capable of
transmitting disease. Disposable gloves must be worn during the assay procedure since
no known test method can offer complete assurance that products derived from human
blood will not transmit infectious agents. Therefore, all blood derivatives should be
considered potentially infectious and good laboratory practices should be followed.
9) All samples should be disposed of in a manner that will inactivate human viruses.
Solid Wastes: Autoclave for 60 minutes at 121°C.
Liquid Wastes: Add sodium hypochlorite to a final concentration of 1.0%. The waste
should be allowed to stand for a minimum of 30 minutes to inactivate viruses before
disposal.
10)Substrate Solution is easily contaminated. If bluish prior to use, do not use.
11)Substrate B contains 20% acetone: Keep this reagent away from sources of heat and
flame.
12)If Wash Buffer (20X) is stored at a lower temperature (2-5°C), crystals may form which
must be dissolved by warming to 37°C prior to use.
9. Preparation

1) Sample preparation

COLLECTION, HANDLING AND STORAGE
a) Cell Culture Supernatant: Centrifuge to remove any visible particulate material.
b) Serum: Blood should be drawn using standard venipuncture techniques and serum
separated from the blood cells as soon as possible. Samples should be allowed to clot
for one hour at room temperature, centrifuged for 10 minutes (4°C), and serum
extracted.
c) Plasma: Blood should be drawn using standard venipuncture techniques and plasma
collected using sodium citrate, EDTA, or heparin as an anticoagulant. To ensure optimal
recovery and minimal platelet contamination, separation of plasma must be done on ice
in less than 30 minutes after collection. Centrifuge for 10 minutes (4°C) to remove any
particulate. This IL-13 Rα2 ELISA kit is not affected by haemolysis of specimens. No
adverse effects have been noted in the presence of anti-coagulants, sodium citrate,
EDTA, or heparin.
• Avoid grossly hemolytic, lipidic or turbid samples.
• Serum, plasma, and cell culture supernatant to be used within 24-48 hours may be
stored at 2-8°C, otherwise samples must stored at -20°C to avoid loss of bioactivity and
contamination. Avoid freeze-thaw cycles.
• When performing the assay, slowly bring samples to room temperature.
• It is recommended that all samples be assayed in duplicate.
• DO NOT USE HEAT-TREATED SPECIMENS.
2) Preparation of Reagent
Remove all kit reagents from refrigerator and allow them to reach room temperature (20-
25°C). Prepare the following reagents as indicated below. Mix thoroughly by gently
swirling before pipetting. Avoid foaming.
1. Wash Buffer (1X): Add 60 mL of Wash Buffer (20X) and dilute to a final volume of

1200 mL with distilled or deionized water. Mix thoroughly. If a smaller volume of Wash
Buffer (1X) is desired, add 1 volume of Wash Buffer (20X) to 19 volumes of distilled or
deionized water. Wash Buffer (1X) is stable for 1 month at 2-8°C. Mix well before use.
2. Substrate Solution: Substrate A and Substrate B should be mixed together in equal
volumes up to 15 minutes before use. Refer to the table provided for correct amounts of
Substrate Solution to prepare.
Strips Used Substrate A (mL) Substrate B (mL) Substrate Solution (mL)
2 strips (16 wells) 2.0 2.0 4.0
4 strips (32 wells) 3.0 3.0 6.0
6 strips (48 wells) 4.0 4.0 8.0
8 strips (64 wells) 5.0 5.0 10.0
10 strips (80 wells) 6.0 6.0 12.0
12 strips (96 wells) 7.0 7.0 14.0
3. IL-13 Rα2 Standard:
a) Two vials of Standard are provided in this package to allow both serum/plasma and cell
culture supernatant testing. Reconstitute IL-13 Rα2 Standard with 2.0 ml of Calibrator
Diluent I or Calibrator Diluent II. This reconstitution produces a stock solution of 8
ng/ml. Allow solution to sit for at least 15 minutes with gentle agitation prior to making
dilutions. Use within one hour of reconstituting. The IL-13 Rα2 standard stock solution
can be stored frozen (-20°C) for up to 10 days. Avoid freeze-thaw cycles: aliquot if
repeated use is expected.
b) Use the above stock solution to produce a serial 2-fold dilution series, as described elow,
within the range of this assay (100 pg/mL to 6.4 ng/mL) as illustrated. Add 0.5 ml of the
appropriate Calibrator Diluent to each test tube. Between each test tube transfer, be sure
to mix contents thoroughly. The undiluted IL-13 Rα2 stock solution will serve as the
high standard (6.4 ng/mL) and the Calibrator Diluent will serve as the zero standard (0
ng/mL).

10. Assay Procedure
1) Prepare Wash Buffer (1X) and IL-13Ra2 Standards before starting assay procedure (see
Preparation of Reagents). It is recommended that the table and diagram provided be
used as a reference for adding Standards or Samples to the Microtiter Plate.
Wells Contents Wells Contents
1A, 1B Standard 1 - 0 pg/mL (S1) 2C, 2D Standard 6 – 1.6 ng/mL (S6)
1C, 1D Standard 2 – 100 pg/mL (S2) 2E, 2F Standard 7 – 3.2 ng/mL (S7)
1E, 1F Standard 3 – 200 pg/mL (S3) 2G, 2H Standard 8 – 6.4 ng/mL (S8)
1G, 1H Standard 4 – 400 pg/mL (S4) 3A-12H IL-13 Rα2 samples
2A, 2B Standard 5 – 800 pg/mL (S5)
1 2 3 4 5 6 7 8 9 10 11 12
A S1 S5 1 5 9 13 17 21 25 29 32 36
B S1 S5 1 5 9 13 17 21 25 29 32 37
C S2 S6 2 6 10 14 18 22 26 30 33 37
D S2 S6 2 6 10 14 18 22 26 30 33 38
E S3 S7 3 7 11 15 19 23 27 31 34 39
F S3 S7 3 7 11 15 19 23 27 31 34 39
G S4 S8 4 8 12 16 20 24 28 32 35 40
H S4 S8 4 8 12 16 20 24 28 32 35 40
2) Add 100 ul of Standard or Sample to the appropriate well of the antibody pre-coated
Microtiter Plate and incubate 1 hour at 37℃.
3) Without discarding the standards and samples, add 50 ul of IL-13 Rα2 Biotin conjugate
to each wells. Mix well. Cover and incubate for 1 hour at 37℃.
4) Wash the Microtiter Plate using one of the specified methods indicated below:
Manual Washing: Remove incubation mixture by aspirating contents of the plate into a
sink or proper waste container. Using a squirt bottle, fill each well completely with Wash
Buffer (1X) then aspirate contents of the plate into a sink or proper waste container.
Repeat this procedure four more times for a total of FIVE washes. After final wash,
invert plate, and blot dry by hitting plate onto absorbent paper or paper towels until no
moisture appears. Note: Hold the sides of the plate frame firmly when washing the plate
to assure that all strips remain securely in frame.
Automated Washing: Aspirate all wells, then wash plates FIVE times using Wash
Buffer (1X). Always adjust your washer to aspirate as much liquid as possible and set fill
volume at 350 ul/well/wash (range: 350-400 ul). After final wash, invert plate, and blot

dry by hitting plate onto absorbent paper or paper towels until no moisture appears. It is
recommended that the washer be set for a soaking time of 10 seconds or shaking time of
5 seconds between washes.
5) Dispense 100 ul of Avidin Conjugate to each well Mix well. Cover and incubate for 1
hour at 37℃.
6) Prepare Substrate Solution no more than 15 minutes before end of second incubation
(see Preparation of Reagents).
7) Repeat wash procedure as described in Step 4.
8) Add 100 ul Substrate Solution to each well. Cover and incubate for 15-20 minutes at
37℃.
9) Add 100 ul Stop Solution to each well. Mix well.
10) Read the Optical Density (O.D.) at 450 nm using a microtiter plate reader set within 30
minutes.
11. Characteristics
1) Calculation of results
The standard curve is used to determine the amount of IL-13 Rα2 in an unknown sample.
The standard curve is generated by plotting the average O.D. (450 nm) obtained for each
of the standard concentrations on the vertical (Y) axis versus the corresponding IL-13
Rα2 concentration (ng/mL) on the horizontal (X) axis.
1. First, calculate the mean O.D value for each standard and sample. All O.D. values are
subtracted by the value of the zero-standard (0 ng/mL) or (S1) before result
interpretation. Construct the standard curve using graph paper or statistical software.
2. To determine the amount of IL-13 Rα2 in each sample, first locate the O.D. value on the
Y-axis and extend a horizontal line to the standard curve. At the point of intersection,
draw a vertical line to the X-axis and read the corresponding IL-13 Rα2 concentration.
If samples have been diluted, the concentration read from the standard curve must be
multiplied by the dilution factor.
3. If samples generate values higher than the highest standard, dilute the samples and
repeat the assay.
2) Typical data
Results of a typical standard run of an IL-13 Rα2 ELISA are shown below. Any variation
in standard diluent, operator, pipetting and washing technique, incubation time or
temperature, and kit age can cause variation in result. The following examples are for the
purpose of illustration only, and should not be used to calculate unknowns. Each user

should obtain its own result.
EXAMPLE
The following data was obtained for a standard curve using Calibrator Diluent II.
Standard (pg/mL) Mean O.D. (450 nm) %CV Zero Standard Subtracted (Std.) - (S1)
0 0.0945 6.73 0.000
100 0.1335 0.53 0.039
200 0.1640 0.00 0.069
400 0.2245 2.20 0.130
800 0.3470 5.60 0.253
1600 0.6290 4.92 0.535
3200 1.0850 3.13 0.991
6400 1.9260 2.73 1.831
3) SENSITIVITY
The sensitivity of human IL-13 Ra as observed by the standard curve generated for both
Calibratory Diluent 1 and Calibrator Diluent II is about 25pg/ml. It is defined as the
detected quantity 2SD from the mean OD of 16 replicates of the zero standard.
4) SPECIFICITY
This sandwich ELISA recognises both natural and recombinant human IL-13 Ra2. This
kit has been tested and exhibited no significant cross-reactivity with following cytokines
and growth factors: IL-1α, IL-1β, IL-2, IL-4, IL-5, Il-6, IL-7, IL-8, IL-10, IL-11, Il-12, Il17A, FGF basic, GM-CSF, IFN-γ, M-CSF, MCP-1(MCAF), MCP3, EGF, TNF- α, TNF-β

12. Troubleshooting
Problem Possible Cause Solution
High signal and background
in all wells
• Insufficient washing • Increase number of washes
• Increase time of soaking
between in wash
• Too much AV-HRP • Check dilution, titration
• Incubation time too long • Reduce incubation time
• Development time too long • Decrease the incubation time
before the stop solution is
added
No signal
• Reagent added in incorrect
order, or incorrectly prepared
• Review protocol
• Standard has gone bad
(If there is a signal in the
sample wells)
• Check the condition of stored
standard
• Assay was conducted from
an incorrect starting point
• Reagents allows to come to
20~30 before performing ℃
assay
Too much signal – whole
plate turned uniformly blue
• Insufficient washing
– unbound AV-HRP remaining
• Increase number of washes
carefully
• Too much AV-HRP • Check dilution
• Plate sealer or reservoir
reused, resulting in presence
of residual AV-HRP
• Use fresh plate sealer and
reagent reservoir for each
step
Standard curve achieved but
poor discrimination between
point
• Plate not developed long
enough
• Increase substrate solution
incubation time
• Improper calculation of
standard curve dilution
• Check dilution, make new
standard curve
No signal when a signal is
expected, but standard curve
looks fine
• Sample matrix is masking
detection
• More diluted sample
recommended
Samples are reading too high,
but standard curve is fine
• Samples contain protein
levels above assay range
• Dilute samples and run
again
Edge effect
• Uneven temperature around
work surface
• Avoid incubating plate in
areas where environmental
conditions vary
• Use plate sealer

13. Reference
1) Biswajit Roy et al. IL-13 signal transduction in human monocytes: phosphorylation of
receptor components, association with Jaks, and phosphorylation/activation of Stats.
Journal of Leukocyte Biology. 2002; Vol. 72: 580-589
2) Brigitte Granel et al. IL-13Ra2 gene polymorphisims are associated with systemic
sclerosis. The Journal of Rheumatology. 2006; Vol. 33 102015-2019
3) Edwin W Lai, Bharat H Joshi, Lucia Martiniova et al. Overexpression of interleukin-13
receptor 2 in neuroendocrine malignant pheochromocytoma: A novel target for
Receptor directed anti-cancer therapy. The Journal of Clinical Endocrinology &
Metabolism. 2009; Vol. 94, No. 8 2952-2957
4) Makoto Saka et al. Vaccine therapy with dendritic cells transfected with IL13 Ra2
mRNA for glima in mice. The Journal of Neurosurgery. 2010; Vol. 113(2):270-9.
5) Mitomu Kioi et al. Interleukin-13 receptor alpha2 chain: a potential biomarker and
molecular target for ovarian cancer therapy. Cancer 2006; Vol. 107(6): 1407–18

◈ Ordering Information

For orders, please contact :
Young In Frontier Co., Ltd.

E-mail: orders@younginfrontier.com
Address: 11F, Byucksan Digital Valley 5th, Gasan-dong 60-73, Geumcheon-gu, Seoul,
Korea (153-788)
Website: http://www.abfrontier.com
Or, your local distributor.
For technical advice, please contact:
E-mail : orders@younginfrontier.com
Website : http://www.abfrontier.com