Quantikine
Human uPAR Immunoassay
Catalog Number DUP00
For the quantitative determination of human urokinase-type
plasminogen activator receptor （uPAR） concentrations in cell
culture supernates, serum, plasma, and urine.
This package insert must be read in its entirety before using this product.
FOR RESEARCH USE ONLY.
NOT FOR USE IN DIAGNOSTIC PROCEDURES.
TABLE OF CONTENTS
Contents Page
INTRODUCTION 2
PRINCIPLE OF THE ASSAY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
LIMITATIONS OF THE PROCEDURE 3
MATERIALS PROVIDED . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
STORAGE 4
OTHER SUPPLIES REQUIRED . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
PRECAUTION 4
SAMPLE COLLECTION AND STORAGE . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
SAMPLE PREPARATION 5
REAGENT PREPARATION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
ASSAY PROCEDURE 7
ASSAY PROCEDURE SUMMARY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
CALCULATION OF RESULTS 9
TYPICAL DATA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
TECHNICAL HINTS 10
PRECISION. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
LINEARITY 11
SENSITIVITY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
CALIBRATION 11
SAMPLE VALUES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
SPECIFICITY 13
REFERENCES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
PLATE LAYOUT 15
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INTRODUCTION
Urokinase-type plasminogen activator receptor （uPAR, CD87） is a cell surface receptor that
binds urokinase-type plasminogen activator （uPA） with high affinity, thereby facilitating the
pericellular activation of plasminogen （see references 1 and 2 for reviews）. uPAR, uPA and
plasminogen activator inhibitor-1 （PAI-1）, form a triad with multiple functions, including
regulation of cell attachment, migration, proliferation and differentiation, by both proteolytic and
nonproteolytic mechanisms （2）.
uPAR is anchored to extracellular surfaces through a glycosyl phosphatidylinositol （GPI）
linkage, with no transmembrane domain （3）. uPAR is synthesized as a 335-amino-acid
polypeptide that includes a 22-residue signal peptide （4）. A 30-residue peptide is cleaved from
the C-terminus during addition of the GPI anchor （3）. With loss of the signal peptide and the
C-terminal peptide, the mature protein has 283 amino acids. It is variably glycosylated,
increasing its mass from about 31 kDa for the protein backbone to as much as 55 kDa for the
mature glycoprotein （5）.
Pro-uPA, a single-chain protein, is activated to a disulfide-linked, two-chain protein by
proteolytic cleavage by plasmin （1, 2, 6）. Either pro-uPA or the active two-chain uPA bind with
high affinity to uPAR. Thus, traces of plasmin activate pro-uPA to uPA, leading to increasing
generation of plasmin in a positive feedback loop that is amplified by uPAR. While the initiating
event is not clear, the effect is the generation of plasmin at the cell surface, where it degrades
the extracellular matrix by activating matrix metalloproteinases. This appears to be a key event
in tumor invasiveness and metastasis and in migration of cells in general （1, 2, 7）.
The functions of the uPA/uPAR system are, however, more extensive than mediation of
plasmin formation, and they include non-proteolytic functions （1, 2）. uPA/uPAR is localized to
focal contact points of cells within the substratum. These sites include intracellular vinculin and
the extracellular adhesion molecule vitronectin, suggesting direct adhesive functions and
intracellular signalling functions for uPAR. uPAR binds to integrins, and it contains chemotactic
activity in its single protease-sensitive region.
uPAR has been measured in human plasma （7 - 9）. Soluble uPAR is generated by removal of
the GPI anchor by an endogenous phospholipase D, freeing uPAR of its surface attachment
（10）. uPAR is elevated in plasma of patients with paroxysmal nocturnal hemoglobinuria （7, 8）,
a manifestation of an inability to add GPI anchors to proteins. It has been postulated that there
also may be soluble uPAR due to alternative splicing of the primary transcript （1）, as has been
demonstrated for mouse uPAR （11）. uPAR has been identified in urine, where the level is a
consistent fraction of creatinine concentration （12）.
The Quantikine Human uPAR Immunoassay is a 4.5 hour solid-phase ELISA designed to
measure human uPAR in cell culture supernates, serum, plasma, and urine. It contains
NS0-expressed recombinant human uPAR and antibodies raised against the recombinant
factor. It has been shown to accuray quantitate the recombinant factor. Results obtained
using natural human uPAR showed linear curves that were parallel to the standard curves
obtained using the Quantikine kit standards. These results indicate that this kit can be used to
determine relative mass values of natural human uPAR.
2
PRINCIPLE OF THE ASSAY
This assay employs the quantitative sandwich enzyme immunoassay technique. A monoclonal
antibody specific for uPAR has been pre-coated onto a microplate. Standards and samples are
pipetted into the wells and any uPAR present is bound by the immobilized antibody. After
washing away any unbound substances, an enzyme-linked polyclonal antibody specific for
uPAR is added to the wells. Following a wash to remove any unbound antibody-enzyme
reagent, a substrate solution is added to the wells and color develops in proportion to the
amount of uPAR bound in the initial step. The color development is stopped and the intensity
of the color is measured.
LIMITATIONS OF THE PROCEDURE
FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES.
The kit should not be used beyond the expiration date on the kit label.
Do not mix or substitute reagents with other lots or sources.
If samples fall outside the dynamic range of the assay, further dilute the samples with
Calibrator Diluent and repeat the assay.
Any variation in standard diluent, operator, pipetting technique, washing technique,
incubation time or temperature, and kit age can cause variation in binding.
This assay is designed to eliminate interference by ligands and other proteins present in
biological samples. Until all factors have been tested in the Quantikine Immunoassay, the
possibility of interference cannot be excluded.
MATERIALS PROVIDED
uPAR Microplate （Part 890714） - 96 well polystyrene microplate （12 strips of 8 wells） coated
with a mouse monoclonal antibody against uPAR.
uPAR Conjugate （Part 890715） - 21 mL of polyclonal antibody against uPAR conjugated to
horseradish peroxidase with preservatives.
uPAR Standard （Part 890716） - 40 ng of recombinant human uPAR in a buffered protein base
with preservatives; lyophilized.
Assay Diluent RD1W （Part 895117） - 11 mL of a buffered protein base with preservatives.
Calibrator Diluent RD6P （Part 895118） - 21 mL of animal serum with preservatives.
Wash Buffer Concentrate （Part 895003） - 21 mL of a 25-fold concentrated solution of
buffered surfactant with preservatives.
Color Reagent A （Part 895000） - 12.5 mL of stabilized hydrogen peroxide.
Color Reagent B （Part 895001） - 12.5 mL of stabilized chromogen （tetramethylbenzidine）.
Stop Solution （Part 895032） - 6 mL of 2 N sulfuric acid.
Plate Covers - 4 adhesive strips.
3
STORAGE
Unopened Kit Store at 2 - 8° C. Do not use past kit expiration date.
Opened/
Reconstituted
Reagents
Diluted Wash Buffer
May be stored for up to 1 month at 2 - 8° C.*
Stop Solution
Assay Diluent RD1W
Calibrator Diluent RD6P
Conjugate
Unmixed Color Reagent A
Unmixed Color Reagent B
Standard
Microplate Wells
Return unused wells to the foil pouch containing the
desiccant pack, reseal along entire edge of zip-seal.
May be stored for up to 1 month at 2 - 8° C.*
*Provided this is within the expiration date of the kit.
OTHER SUPPLIES REQUIRED
Microplate reader capable of measuring absorbance at 450 nm, with the correction
wavelength set at 540 nm or 570 nm.
Pipettes and pipette tips.
Deionized or distilled water.
Squirt bottle, manifold dispenser, or automated microplate washer.
500 mL graduated cylinder.
Test tubes for dilution.
Human uPAR Controls （optional; available from R&D Systems）.
PRECAUTION
The Stop Solution provided with this kit is an acid solution. Wear eye, hand, face, and clothing
protection when using this material.
4
SAMPLE COLLECTION AND STORAGE
Cell Culture Supernates - Remove particulates by centrifugation and assay immediay or
aliquot and store samples at -20° C. Avoid repeated freeze-thaw cycles.
Serum - Use a serum separator tube （SST） and allow samples to clot for 30 minutes before
centrifugation for 10 minutes at 1000 x g. Remove serum and assay immediay or aliquot and
store samples at -20° C. Avoid repeated freeze-thaw cycles.
Plasma - Collect plasma using heparin or EDTA as an anticoagulant. Centrifuge at 1000 x g
within 30 minutes of collection. Assay immediay or aliquot and store samples at -20° C.
Avoid repeated freeze-thaw cycles.
Note: Citrate plasma has not been validated for use in this assay. Lipemic samples and
samples containing high levels of human serum albumin are not suitable for the measurement
of human uPAR with this assay.
Urine - Aseptically collect the first urine of the day （mid-stream）, voided directly into a sterile
container. Centrifuge to remove particulate matter. Assay immediay or aliquot and store at
-20° C. Avoid repeated freeze-thaw cycles. For more information on collecting and handling
urine specimens, refer to the National Committee for Clinical Laboratory Standards （NCCLS）
publication GP16-T.
SAMPLE PREPARATION
Cell culture supernates, serum, plasma, and urine samples require at least a 5-fold dilution. A
suggested 5-fold dilution is 25 L of sample + 100 L of Calibrator Diluent RD6P.
5
REAGENT PREPARATION
Bring all reagents to room temperature before use.
Wash Buffer - If crystals have formed in the concentrate, warm to room temperature and mix
gently until the crystals have compley dissolved. Dilute 20 mL of Wash Buffer Concentrate
into deionized or distilled water to prepare 500 mL of Wash Buffer.
Substrate Solution - Color Reagents A and B should be mixed together in equal volumes
within 15 minutes of use. Protect from light. 200 L of the resultant mixture is required per well.
uPAR Standard - Reconstitute the uPAR Standard with 1.0 mL of deionized or distilled water.
This reconstitution produces a stock solution of 40,000 pg/mL. Allow the standard to sit for a
minimum of 15 minutes with gentle agitation prior to making dilutions.
Pipette 450 L of Calibrator Diluent RD6P into the 4000 pg/mL tube. Pipette 200 L of
Calibrator Diluent RD6P into the remaining tubes. Use the stock solution to produce a dilution
series （below）. Mix each tube thoroughly before the next transfer. The 4000 pg/mL standard
serves as the high standard. Calibrator Diluent RD6P serves as the zero standard （0 pg/mL）.
6
ASSAY PROCEDURE
Bring all reagents and samples to room temperature before use. It is recommended
that all samples, standards, and controls be assayed in duplicate.
1. Prepare all reagents, working standards, and samples as directed in the previous
sections.
2. Remove excess microplate strips from the plate frame, return them to the foil pouch
containing the desiccant pack, and reseal.
3. Add 100 L of Assay Diluent RD1W to each well.
4. Add 50 L of Standard, control, or sample* per well. Cover with the adhesive strip
provided. Incubate for 2 hours at room temperature. A plate layout is provided to record
standards and samples assayed.
5. Aspirate each well and wash, repeating the process three times for a total of four washes.
Wash by filling each well with Wash Buffer （400 L） using a squirt bottle, manifold
dispenser, or autowasher. Complete removal of liquid at each step is essential to good
performance. After the last wash, remove any remaining Wash Buffer by aspirating or
decanting. Invert the plate and blot it against clean paper towels.
6. Add 200 L of uPAR Conjugate to each well. Cover with a new adhesive strip. Incubate
for 2 hours at room temperature.
7. Repeat the aspiration/wash as in step 5.
8. Add 200 L of Substrate Solution to each well. Incubate for 30 minutes at room
temperature. Protect from light.
9. Add 50 L of Stop Solution to each well. The color in the wells should change from blue to
yellow. If the color in the wells is green or if the color change does not appear uniform,
gently tap the plate to ensure thorough mixing.
10. Determine the optical density of each well within 30 minutes, using a microplate reader set
to 450 nm. If wavelength correction is available, set to 540 nm or 570 nm. If wavelength
correction is not available, subtract readings at 540 nm or 570 nm from the readings at
450 nm. This subtraction will correct for optical imperfections in the plate. Readings made
directly at 450 nm without correction may be higher and less accurate.
*Samples require dilution. See Sample Preparation section.
7
ASSAY PROCEDURE SUMMARY
8
CALCULATION OF RESULTS
Average the duplicate readings for each standard, control, and sample and subtract the
average zero standard optical density.
Create a standard curve by reducing the data using computer software capable of generating a
log/log curve-fit. As an alternative, construct a standard curve by plotting the mean absorbance
for each standard on the y-axis against the concentration on the x-axis and draw a best fit
curve through the points on a log/log graph. The data may be linearized by plotting the log of
the uPAR concentrations versus the log of the O.D. on a linear scale, and the best fit line can
be determined by regression analysis.
If the samples have been diluted, the concentration read from the standard curve must be
multiplied by the dilution factor.
TYPICAL DATA
This standard curve is provided for demonstration only. A standard curve should be generated
for each set of samples assayed.
9
pg/mL
0
62.5
125
250
500
1000
2000
4000
O.D.
0.046
0.044
0.072
0.073
0.105
0.105
0.170
0.171
0.300
0.304
0.565
0.545
1.066
1.075
2.122
2.076
Average
0.045
0.072
0.105
0.170
0.302
0.555
1.070
2.099
Corrected
___
0.027
0.060
0.125
0.257
0.510
1.025
2.054
TECHNICAL HINTS
When mixing or reconstituting protein solutions, always avoid foaming.
To avoid cross-contamination, change pipette tips between additions of each standard
level, between sample additions, and between reagent additions. Also, use separate
reservoirs for each reagent.
When using an automated plate washer, adding a 30 second soak period following the
addition of wash buffer, and/or rotating the plate 180 degrees between wash steps may
improve assay precision.
To ensure accurate results, proper adhesion of plate sealers during incubation steps is
necessary.
Substrate Solution should remain colorless until added to the plate. Keep the Substrate
Solution protected from light. Substrate Solution should change from colorless to
gradations of blue.
Stop Solution should be added to the plate in the same order as the Substrate Solution.
The color developed in the wells will turn from blue to yellow upon addition of the Stop
Solution. Wells that are green in color indicate that the Stop Solution has not mixed
thoroughly with the Substrate Solution.
PRECISION
Intra-assay Precision （Precision within an assay）
Three samples of known concentration were tested twenty times on one plate to assess
intra-assay precision.
Inter-assay Precision （Precision between assays）
Three samples of known concentration were tested in forty separate assays to assess
inter-assay precision.
Intra-assay Precision Inter-assay Precision
Sample 1 2 3 1 2 3
n 20 20 20 40 40 40
Mean
（pg/mL）
836 1593 2412 796 1546 2300
Standard
deviation
17.3 65.4 181 44.3 78.9 136
CV （%） 2.1 4.1 7.5 5.6 5.1 5.9
10
LINEARITY
To assess the linearity of the assay, samples spiked with high concentrations of uPAR were
diluted with Calibrator Diluent RD6P to produce samples with values within the dynamic range
of the assay.
Cell culture
media*
（n = 4）
Serum*
（n = 5）
Heparin
plasma*
（n = 5）
EDTA
plasma*
（n = 5）
Urine*
（n = 5）
1:2
Average % of Expected 103
96 - 112
104
101 - 105
104
101 - 106
101
94 - 105
102
Range （%） 99 - 105
1:4
Average % of Expected 109
101 - 115
106
103 - 111
107
104 - 110
105
101 - 108
104
Range （%） 95 - 112
1:8
Average % of Expected 109
101 - 111
106
103 - 112
106
102 - 110
106
103 - 113
108
Range （%） 104 - 113
1:16
Average % of Expected 109
99 - 114
105
99 - 111
105
98 - 110
104
98 - 111
108
Range （%） 103 - 112
*Samples were diluted 5-fold prior to assay as directed in Sample Preparation.
SENSITIVITY
The minimum detectable dose （MDD） of uPAR is typically less than 33 pg/mL.
The MDD was determined by adding two standard deviations to the mean optical density value
of twenty zero standard replicates and calculating the corresponding concentration.
CALIBRATION
This immunoassay is calibrated against a highly purified NS0-expressed recombinant human
uPAR produced at R&D Systems.
11
SAMPLE VALUES
Serum/Plasma/Urine - Samples from apparently healthy volunteers were evaluated for the
presence of uPAR in this assay. No medical histories were available for the donors used in this
study.
Sample Type Mean （pg/mL） Range （pg/mL）
Serum （n = 60） 2370 1195 - 4415
Heparin plasma （n = 35） 2165 977 - 5347
EDTA plasma （n = 35） 1871 864 - 3829
Urine （n = 35） 2975 691 - 6098
Cell Culture Supernates - Human peripheral blood mononuclear cells （5 x 106 cells/mL）
were cultured in RPMI supplemented with 5% fetal calf serum, 50 M -mercaptoethanol,
2 mM L-glutamine, 100 U/mL penicillin, and 100 g/mL streptomycin sulfate. The cells were
cultured unstimulated or stimulated with 10 g/mL PHA. Aliquots of the cell culture supernates
were removed and assayed for levels of natural uPAR.
Condition Day 1 （pg/mL） Day 5 （pg/mL）
Unstimulated 1228 2123
Stimulated 10,005 6895
12
SPECIFICITY
This assay recognizes recombinant and natural human uPAR. The factors listed below were
prepared at 50 ng/mL in Calibrator Diluent RD6P and assayed for cross-reactivity.
Preparations of the following factors at 50 ng/mL in a mid-range recombinant human uPAR
control were assayed for interference. No significant cross-reactivity or interference was
observed.
Recombinant human uPA does not cross-react in this assay but does interfere at
concentrations greater than 3000 pg/mL.
13
Recombinant
human:
ANG
AR
CNTF
-ECGF
EGF
Epo
FGF acidic
FGF basic
FGF-4
FGF-5
FGF-6
Flt-3 Ligand
G-CSF
GM-CSF
sgp130
GRO
GRO
GRO
HB-EGF
HGF
IFN-
IGF-I
IGF-II
IL-1
IL-1
IL-1ra
IL-1 sRI
IL-1 sRII
IL-2
IL-2 sR
IL-3
IL-3 sR
IL-4
IL-4 sR
IL-5
IL-5 sR
IL-5 sR
IL-6
IL-6 sR
IL-7
IL-8
IL-9
IL-10
IL-11
IL-12
IL-13
KGF （FGF-7）
LAP （TGF-1）
LIF
M-CSF
MCP-1
MIP-1
MIP-1
-NGF
OSM
PD-ECGF
PDGF-AA
PDGF-AB
PDGF-BB
PTN
RANTES
SCF
SLPI
TGF-
TGF-1
TGF-2
TGF-3
TGF- sRII
TNF-
TNF-
sTNF RI
sTNF RII
VEGF
Recombinant
mouse:
GM-CSF
IL-1
IL-1
IL-3
IL-4
IL-5
IL-6
IL-7
IL-9
IL-10
IL-13
LIF
MIP-1
MIP-1
SCF
TNF-
uPAR
Recombinant
amphibian:
TGF-5
Natural
proteins:
bovine FGF acidic
bovine FGF basic
human PDGF
porcine PDGF
human TGF-1
porcine TGF-1
REFERENCES
1. Dear, A.E. and R.L. Medcalf （1998） Eur. J. Biochem. 252:185.
2. Blasi, F. （1997） Immunlogy Today 18:415.
3. Ploug, M. et al. （1991） J. Biol. Chem. 266:1926.
4. Roldan, A.L. （1990） EMBO J. 9:467.
5. Behrendt, N. （1990） J. Biol. Chem. 265:6453.
6. Blasi, F. et al. （1987） J. Cell Biol. 104:801.
7. Ronne, E. et al. （1995） Br. J. Haematol. 89:576.
8. Ploug, M. et al. （1992） Blood 79:1447.
9. Stephens, R.W. et al. （1999） J. Natl. Cancer Inst. 91:869.
10. Wilhelm, O.G. et al. （1999） J. Cellular Physiol. 180:225.
11. Kristensen, P. et al. （1991） J. Cell Biol. 115:1763.
12. Sier, C.F.M. et al. （1999） Lab. Invest. 79:717.
14
PLATE LAYOUT
Use this plate layout to record standards and samples assayed.
© 2010 R&D Systems, Inc.
12.99 750440.5 7/10