HUMAN SOLUBLE INTERLEUKIN-2 RECEPTOR

Wednesday, May 05, 2004

Description
IL-2 receptor alpha chain is one of the key components of the lymphoid
cell receptor complex. Interleukin-2(IL-2), plays a pivotal role in
initiating and potentiating the immune response. It provides a critical
signal necessary for both T Cell proliferation and antibody secretion by
B cells. In fact, IL-2 has a wide range of effects upon many lymphoid
cells that express IL-2 receptor including natural killer cells and
monocytes. Following stimulation by antigen, all T cells express highaffinity
IL-2 receptors, while those with a T helper phenotype transiently
secrete IL-2. The signal provided by the interaction of IL-2 with its
high affinity receptors, composed of a 55-kD "-chain and a 75-kD $-chain,
is necessary and sufficient for T cells to undergo G1 progression, S phase
transition and subsequent cell division.
IL-2 receptor is the protein that mediates the action of IL-2, and normal
T and B cells do not display a significant number of these receptors on
their surface. Stimulation of the immune system causes two IL-2R "
changes:
More molecules of IL-2R " are expressed on the cells plasma membrane and
the soluble form of the IL-2 Receptor alpha is released by the activated
cells into the surrounding fluid.
The principle of the CELLFREE Human s-IL-2R test kit is an
enzymeimmunoassay (EIA) based on the sandwich principle. It is for
determination of soluble Interleukin-2 receptor (IL-2R) levels in human
serum, plasma,or cell culture supernatant.
A monoclonal antibody specific for sIL-2R has been coated onto the
microtiter plate provided in the kit. Samples and standards are pipetted
into the wells of the microtiter plate.
Immediately enzyme conjugated anti-IL-2R monoclonal antibody is added into
micotiter wells. The sIL-2R present in the standards or samples binds to
the coated antibody while the conjugated antibody binds to a second,
distinct epitope on the IL-2R molecule completing the sandwich. After
incubation the unbound material present in the sample is removed by
washing. Next, a chromogen solution is added to the wells forming a
colored end product that is proportional to the amount of IL-2R present in
the samples.
The enzyme reaction is stopped by the addition of Stop solution and the
absorbance at 490 nm is measured with a spectrophotometer.
A standard curve is obtained by plotting the optical density (absorbances)
versus the corresponding concentrations of defined standards. The human
sIL-2R concentration of samples with unknown concentrations, which are run
concurrently with the Standards, can be determined from the standard
curve.
sIL-2R is normally measurable in plasma, serum, and other body fluids, and
the concentration rises in inflammatory and noninflammatory diseases. Any
pathological factor which stimulate T-Cells and other cells causes
increase in sIL-2R in the circulation.
sIL-2R is a stable marker of immune activation in biologic fluids and
cannot be rapidly cleared from the circulation.
Increased concentrations of the sIL-2R are seen in the majority of
individuals with human immunodeficiency virus (HIV) infection and other
diseases characterized by immune activation (Breast cancer, Multiple
Sclerosis, rheumatoid arthritis, etc.). The assay is often described as
a useful parameter for evaluating diseases activity, diseases prognosis
and evaluation of immune system.

Procedure
SPECIMENS:
2.1 Patient preparation:
There are no specific instructions for the patient, but it is
preferable to avoid fatty food for a at least two hours before blood
draw.
2.2 Specimen Collection:
2.2.1 Blood Collection:
2.2.1.1 Serum:
- Blood is collected by venipuncture,
according to approved procedure for
collection of diagnostic blood specimens by
venipuncture, into a serum collecting tube
(red-top vacutainer). Allow specimen to
clot (0, 5,-1 hour) at room temperature.
- Separate the serum by centrifugation at 500
x g for ten minutes.
- Aliquot the serum into 1.8 ml labeled
cryovials.
- Minimum amount of 100 l and maximum amount
of 500 l of serum is required for sIL-2
receptor assay.
- If more than 2 hours elapses before
separation, place the tube at 4EC in the
refrigerator.
2.2.1.2 Plasma:
- Blood collected by venipuncture using an
appropriate anticoagulant (EDTA, Heparin or
Citrate).
- Mix the specimen gently and allow the tube
to stand for 1/2 hour.
- Separate the plasma from the other
components of the blood by centrifugation
(350 x g for 15 minutes).
- Aliquot 1.8 ml of plasma into 1.8 ml
labeled cryovial.
- Minimum amount of 100 l and the maximum
amount of 500 l of plasma is required for
sIL-2 receptor assay.
NOTE: Hemolyzed samples and lipemic samples may
interfere with the determination of sIL-2R. If
possible, another sample should be drawn. The
results of hemolyzed and lipemic samples may be
reported but should be noted.
2.2.2 Culture Supernatants:
- record PID, Stimulant, Time, and Date of harvest of each
well and labeled all tubes are to be used with alcohol
resistant marker.
- Pipette out cells free supernatants from the wells and
add into labeled tube according to your protocol.
- All supernatants from same stimuli should be batch
tested in one assay.
- Minimum sample of 60 l per single determination is
needed for the assay.
- Keep frozen 100 ml of medium used in the cytokine induce
assay for sample dilution if the sIL-2R concentration of
sample exceeds the highest points of the standards
curve.
2.2.3 CSF Collection:
- CSF should be collected into a sterile, screw capped
tube.
- Centrifuge the tube at 1000 x g for ten minutes.
- Pipette CSF out of the tube without disturbing the
pellet, leaving at least 0.2 ml of CSF in the tube.
- Aliquot the CSF into 1.8 ml labeled cryovials and store
at -70EC.
- Minimum amount of 100 l and maximum amount of 500 l
CSF is needed for sIL-2 receptor assay.
- If not processed on-site, store at 4EC and send it to
CIRID.
2.3 Specimen Labeling:
Clearly label the following information on the cryovials:
- PID
- Date of draw
- Type of specimen
2.4 Transport and Storage:
2.4.1 On Site Sample Processing and storage:
- Follow the procedure for your on-site sample collection
and processing.
- Store the samples at -70EC until assay on site or
shipment to central assay Lab.
2.4.2 Ship overnight the frozen samples according to the shipment
rule and regulation for biohazard specimens to central assay
lab.
3. MATERIALS USED IN THE ASSAY:
3.1 Materials provided:
3.1.1 CELLFREE Interleukin-2 Receptor test kit provides as set
sufficient for 96 determinations.
3.2 Materials required but not provided:
3.2.1 Micropipettes and disposable tips (50 l, 100 l, 200 l, and
300 l). For various steps, use of multi-channel pipette (8
channel microwell pipette) is advised.
3.2.2 Volumetric pipettes.
3.2.3 Distilled or deionized water.
3.2.4 Polypropylene tubes.
3.2.5 Linear graph paper
3.2.6 Disposable reagent troughs for multi-channel pipette
3.3 Instrumentation:
3.3.1 Automatic or semi-automatic rinsing/aspiration equipment can
be used to perform the washing steps in the assay procedure.
3.3.2 Titertek Multiskan MCC Microtiter Plate Reader or any other
brand with wavelength set at 450 nm. Clean and Calibrate the
system annually.
3.3.3 Rotating platform 150 10 RPM.
3.3.4 Biosafety Hood: All work done under biosafety hood. Test and
certify the Biosafety hood annually by Technical Safety
Services Inc. 1-(800)-877 7742.
3.3.5 Pipettes: Clean and calibrate pipettes semi-annually by
Calibrate Inc. Pipet Calibration & service 1-(800) 253-7064.
3.4 Reagents & Storage:
3.4.1 1 plate anti-human-IL-2R coated microtiter plate. Each plate
consists of twelve 8-well strips coated with marine monoclonal
antibody to human IL-2R. Store at 2-8EC in the foil bag
provided with desiccant.
3.4.2 1 vial (6 ml) IL-2R Conjugate. The vial contains horseradish
peroxidase (HRP) conjugated marine monoclonal antibody to
human IL-2R in a buffered solution with bovine serum protein
and gentamicin sulfate. Store at 2-8EC. Protect from the
light exposure.
3.4.3 1 vial (6 ml) IL-2R sample Diluent. The vial contains bovine
serum proteins and gentamicin sulfate. Store at 2-8EC.
3.4.4 6 Vials Lyophilized IL-2 R Standards (0.5 ml each). Each vial
contains recombinant human IL-2R in a buffered solution with
bovine-derived serum proteins. Store at 2-8EC. Reconstitute
each vial with 0.5 ml deionized water (see vial for exact U/ml
concentration).
3.4.5 2 vials IL-2R controls. Each vial contains recombinant human
IL-2R in buffered solution with bovine derived serum proteins.
Store at 2-8EC. Reconstitute each vial with 0.5 ml deionized
water (see vial labels for assigned ranges).
3.4.6 Chromogen tablets - 6 tablets, each tablet contains OPhenylenediamine
and fillers.
3.4.7 1 vial (30 ml) Substrate Diluent. A buffered solution
containing urea peroxide and thimerosal. Store at 2-8EC.
3.4.8 1 bottle (50 ml) Wash Buffer concentrate. The bottle contains
20X concentrate buffered solution of detergent. Store the
concentrate at 2-8EC.
3.4.9 2 plastic plate sealers.
3.4.10 Directions for use.4. WARNINGS, LIMITATIONS AND PRECAUTIONS:
4.1 Specimens and all materials coming into contact with the assay
should be handled as if capable of transmitting infection and
disposed of using proper precaution.
4.2 Do not mix reagents from different master lots. Do not use kit
components beyond expiration date.
4.3 Some of the kit may be hazardous to health and direct contact with
skin, eyes etc. should therefore be minimized by careful handling.
The use of gloves and Lab coat during the handling of these
substances and wash hands afterwards is strongly recommended.
4.4 Do not smoke, eat, or drink in the areas where specimens or kit
reagents are handled.
4.5 Do not pipette by mouth.
4.6 Reagents containing Chromogen may be toxic if ingested.
5. ASSAY PROCEDURE:
5.1 General Remarks:
5.1.1 Before performing the assay, samples and assay kit should be
brought to room temperature.
5.1.2 All Standards should be run with each series of unknown
samples.
5.1.3 Each Standard and sample should be assayed in duplicate each
time the test is performed.
5.1.4 Standards should be subject to the same manipulations and
incubation times as the samples being tested.
5.2 Preparation:
5.2.1 Bring samples and reagents to room temperature (18-25EC)
before use. Concentrated Dilution Buffer may contain
crystals. In case crystals do not disappear at room
temperature within 1 hour, concentrated Dilution Buffer can be
warmed up to 37EC; DO NOT SHAKE!
5.2.2 Remove IL-2R microtiter plates from the storage bags and
number the strips to be used with a laboratory marker. To
avoid condensation, do not open the foil pouch containing the
plate until it has reached room temperature. Place strips to
be used in supplied Frame.
5.2.3 Return unused test wells to the storage bag with desiccant,
seal and store at 2-8EC.
5.2.4 Reconstitute the Standards and kit controls with 0.5 ml of
deionized water, swirl gently to mix. After reconstitution,
store at 2-8EC. IF not used again within seven days, store at
-20EC.
5.2.5 Dilute 50 ml of 20X concentrated Wash Buffer (vial 1) to 950
ml with distilled or de-ionized water. In case less tests are
planned, prepare correspondingly less Wash Buffer as required.
Dilute 1 part of the concentrated Wash Buffer (vial 1) with 19
parts distilled or de-ionized water. Mix thoroughly for 15
minutes. Store at 2-8EC for up to 30 days.
5.2.6 Chromogen Solution preparation:
5.2.6.1 Determine the number of Chromogen tablets
required for the assay using the Chromogen
Solution Preparation Chart.
No. of wells No. of Chromogen Volume of Substrate
Tablets diluent
1-40 1 5.0 ml
1-90 2 10.0 ml
1-96 3 15.0 ml
5.2.6.2 Using a clean non-metallic forceps,
transfer the chromogen tablet into a
suitable container.
DO NOT USE A TABLET THAT IS NOT INTACT.
DO NOT USE A TABLET THAT IS YELLOW IN
COLOR.
DO NOT ALLOW TABLETS TO CONTACT YOUR SKIN.
5.2.6.3 Using a clean pipette, transfer 5.0 ml of
substrate diluent per chromogen tablet to
the container. Allow solution to stand a
few minutes, then swirl to dissolve
Chromogen tablet(s). The resultant
solution must be colorless to pale yellow.
A yellow-orange color indicates chromogen
deterioration and the solution should be
discarded.
NOTE: THE CHROMOGEN SOLUTION MUST BE USED
WITHIN 15 MINUTES OF PREPARATION. THIS IS
BEST ACCOMPLISHED BY PREPARING THE WORKING
SOLUTION NO MORE THAN 15 MINUTES PRIOR TO
USE.
5.2.7 Stop Solution (2N H2SO4): Prepare 2N H2SO4 (Acid MUST be
added to water). Store at 2-8EC for up to 6 months.
5.3 Step by Step Human sIL2-R EIA Procedure:
5.3.1 Bring all reagents to room temperature and mix thoroughly
before use without foaming.
5.3.2 Determine the number of strips required to test the desired
number of patient samples plus 22 wells needed for running
Blanks, Standards, and controls (kits and Lab controls).
Remove extra strips from holder and store at 2-8EC in the
resealable foil bag with desiccant.
5.3.3 Leaving the blank wells empty, pipette 50 L of Standards, kit
control, Lab controls and sample in duplicate, into antibody
coated wells according to your scheme.
5.3.4 Leaving the blank wells empty, add 50 L of HRP conjugated
anti-IL-2R antibody to all other wells.
Important: Gently agitate plate by tapping the edge of the
holder for at least 15 seconds to thoroughly mix
contents of each well.
5.3.5 Cover the plate with plate sealer and incubate at room
temperature (24 2EC) for 3 hours on a rotator set at 150
10 RPM.
5.3.6 Approximately 15 minutes before the end of incubation, prepare
Chromogen Solution as directed in 5.2.6.
5.3.7 Remove and discard sealer. Aspirate solution from all wells.
Wash wells six times with approximately 250 L of wash buffer
per well with thorough aspiration between washes.
5.3.8 Pipette 100 L of prepared Chromogen Solution into all wells,
including blank wells. Incubate uncovered for 30 minutes at
room temperature (24 2EC). Do not shake.
5.3.9 Pipette 50 L of the stop solution into all wells, including
blank wells. Tap gently to mix.
NOTE: It is important that Stop Solution be added to wells
prior to reading at 490 nm. Addition of stop Solution
causes an increase in absorbance of the chromogen and a
shift in absorption spectrum.
5.3.10 Place the tray in a spectrophotometer and measure the
absorbance at 490 nm, following the instruction provided
by the instrument manufacturer. The absorbance should be
read as soon as possible after the completion of the
assay, but may be read up to 2 hours after addition of
stop solution when well are kept protected from light at
room temperature.
6. CALCULATION:
6.1 Calculate the average absorbance values (A ) for each set of 490
duplicate Standards, samples and controls.
6.2 If individual absorbance values differ by more than 15% from the
corresponding mean value, the result is considered suspect and the
sample should be re-assayed.
6.3 The mean absorbance value of the zero standard, controls should be
less than 10%
6.4 Construct a standard curve by plotting the mean absorbance for each
Standard on the vertical (Y) axis versus the corresponding sIL-2R
concentration on the horizontal (X) axis.
6.5 Using the mean absorbance value for each sample, determine the
corresponding concentration of sIL-2R from the standard curve. Find
the absorbance value on the Y-axis and extend a horizontal line to
the curve. At the point of intersection, extend a vertical line to
the X-axis and read the sIL-2R concentration for the unknown sample.
6.6 If the samples have been diluted, the concentration determined from
the standard-curve must be multiplied by the dilution factor.
6.7 AssayZap software Windows or Apple versions by (Biosoft Inc.
Tel:(314) 524-8029) is suitable for computer assisted analysis of
sIL2-R.
7. QUALITY CONTROL:
7.1 In accord with modern management practice, quality control and
assurance must be regarded as a collaborative exercise with active
and committed participation of all personnel concerned, from the
most neophyte to the most qualified. Low or zero rejection rates
require corrective actions to be taken before actual problems arise,
which is impossible without the full participation of those most
closely involved. (Precision of pipettes, diluters, readout devices
and good operator technique.)
7.2 The samples used for internal or external quality control purposes
must resemble as closely as possible the patient samples. For this
purpose CIRID at UCLA recommends a large number of aliquots of two
in-house reference samples should be prepared. One set of reference
samples should be normal or slight abnormal and another set should
be distinctly abnormal. The number of aliquots in these separate
quality control pool must be available in sufficient quantity and
sufficient stability for extensive serial laboratory testing in the
lab.
7.3 One aliquot from each of the two In house reference should be used
as an internal laboratory control in each run. Reference samples
should be run in triplicates.
7.4 Kit and In house controls must be routinely assayed as unknown
samples to measure assay variability. The results of all tests
should be serially plotted to monitor the performance of the kits.
7.5 Repeat testing on subsequent assay day of 15-20% patient samples is
also recommended. It used to measure inter-assay precision.
7.6 Proficiency testing:
7.6.1 Participate in a national inter-laboratory proficiency
program, when such a program is developed.
8. EXPECTED VALUES:
Serum/plasma samples from apparently healthy individuals(male and female)
were evaluated for sIL-2R with this assay by the manufacture. The average
concentration of sIL-2R was 529 U/ml and the upper limit was 913 U/ml
(mean + 2 SD) (manufacture data).
8.1 Serum/plasma samples from apparently healthy individuals were
evaluated for sIL-2R with this assay(CIRID at UCLA data)
Sex N Median* Mean* SD* 10th 90th
Male/Female 29 510 539 175 350 740
* U/ml
9. PROCEDURE NOTE:
9.1 The kit should not be used beyond expiration date.
9.2 Since assay condition may vary from assay to assay, a standard curve
must be established for every assay run.
9.3 For values higher than the highest standard should be diluted with
PBS 1X (diluent), and repeat the assay.
9.4 Completely aspirate well contents before dispensing fresh wash
solution.
9.4.1 Fill with wash buffer to the top of the well for each wash
cycle (approx. 250 L).
9.4.2 Do not allow wells to sit uncovered or dry for extended
periods between incubation steps.
9.5 Check the data for:
9.5.1 The Coefficient of Variation (%) of Standards must be less
than 10%.
9.5.2 The Coefficient of Variation (%) of Controls (triplicate) must
be less than 15%.
9.5.3 Random retest of 15-20% of the samples must vary by less than
15% of original value.
9.6 The results are checked and verified by the section supervisor and
reported to the data manager for data entry. Final data print-outs
are checked by the Associate Director or Director of CIRID before
results are sent out.
10. LIMITATION OF THE METHOD:
10.1 This kit is for Research use only. It is not for Diagnosis of
diseases.
10.2 For values higher than the highest standard should be diluted with
sample diluent, and repeat the assay. Recommended dilution is 1:5
(50 l sample 200 L sample diluent).
11. METHOD VALIDATION:
11.1 Minimum Detectable Concentration (MDC): The MDC of the CELLFREE
Interleukin-2 Receptor Test kit is approximately 24 U/ml IL-2R.
11.2 Precision: (manufacture data)
11.2.1 Intra-assay precision was determined from the mean of 20
replicates of each sample:
Sample Mean (U/ml) SD Coefficient
of
Variation %
Serum Pool A 669 39 5.8
Serum pool B 1001 87 8.7
Serum Pool C 3286 150 4.6
11.2.2 Inter-assay precision was determined from the mean of
triplicates of each samples for 20 different assays.
Sample Mean (U/ml) SD CV %
Serum Pool A 703 66 9.4
Serum Pool B 1014 73 7.1
Serum Pool C 3247 246 7.6
11.3 Specificity:
The CELLFREE IL-2R test kit is specific for human IL-2R.
12. GENERAL INFORMATION ABOUT THE PRODUCT:
Name: CELLFREE IL-2R Test Kit
Cat #: CK1025
Manufactured by: T-Cell diagnostics (Endogen)
Distributor: Endogen Inc.
640 Cambridge, MA 02139-4815
Tel: 800-487-4885 (责任编辑：泉水)

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