FOR IN VITRO USE AND RESEARCH USE ONLY
NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES
7th Edition （Revised in November, 2011）
[ INTENDED USE ]
The kit is a sandwich enzyme immunoassay for in vitro quantitative measurement of uPAR in human serum,
plasma and other biological fluids.
[ REAGENTS AND MATERIALS PROVIDED ]
Reagents Quantity Reagents Quantity
Pre-coated, ready to use 96-well strip plate 1 Plate sealer for 96 wells 4
Standard （lyophilized） 2 Standard Diluent 1×20mL
Detection Reagent A （green） 1×120μL Assay Diluent A （2 × concentrate） 1×6mL
Detection Reagent B （red） 1×120μL Assay Diluent B （2 × concentrate） 1×6mL
TMB Substrate 1×9mL Stop Solution 1×6mL
Wash Buffer （30 × concentrate） 1×20mL Instruction manual 1
[ MATERIALS REQUIRED BUT NOT SUPPLIED ]
1. Microplate reader with 450 ± 10nm filter.
2. Precision single or multi-channel pipettes and disposable tips.
3. Eppendorf Tubes for diluting samples.
4. Deionized or distilled water.
5. Absorbent paper for blotting the microtiter plate.
6. Container for Wash Solution
[ STORAGE OF THE KITS ]
1. For unopened kit: All the reagents should be kept according to the labels on vials. The Standard, Detection
Reagent A, Detection Reagent B and the 96-well strip plate should be stored at -20oC upon receipt while the
others should be at 4 oC.
2. For opened kit: When the kit is opened, the remaining reagents still need to be stored according to the above
storage condition. Besides, please return the unused wells to the foil pouch containing the desiccant pack, and
reseal along entire edge of zip-seal.
Note:
It is highly recommended to use the remaining reagents within 1 month provided this is within the expiration date
of the kit.
[ SAMPLE COLLECTION AND STORAGE ]
Serum - Allow samples to clot for two hours at room temperature or overnight at 4oC before centrifugation for 20
minutes at approximay 1000×g. Assay immediay or store samples in aliquot at -20oC or -80oC. Avoid
repeated freeze/thaw cycles.
Plasma - Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples for 15 minutes at
1000×g within 30 minutes of collection. Remove plasma and assay immediay or store samples in aliquot at
-20oC or -80oC. Avoid repeated freeze/thaw cycles.
Other biological fluids - Centrifuge samples for 20 minutes at 1000×g. Remove particulates and assay
immediay or store samples in aliquot at -20oC or -80oC. Avoid repeated freeze/thaw cycles.
Note:
1. Samples to be used within 5 days may be stored at 4oC, otherwise samples must be stored at -20oC （􀀀1
month） or -80oC （􀀀2 months） to avoid loss of bioactivity and contamination.
2. Sample hemolysis will influence the result, so hemolytic specimen can not be detected.
3. When performing the assay, bring samples to room temperature.
[ REAGENT PREPARATION ]
1. Bring all kit components and samples to room temperature （18-25oC） before use.
2. Standard - Reconstitute the Standard with 1.0 mL of Standard Diluent, kept for 10 minutes at room
temperature, shake gently（not to foam）. The concentration of the standard in the stock solution is 200ng/mL.
Please firstly dilute the stock solution to 20ng/mL and the diluted standard serves as the highest standard
（20ng/mL）. Then prepare 7 tubes containing 0.5mL Standard Diluent and use the diluted standard to produce
a double dilution series according to the picture shown below. Mix each tube thoroughly before the next
transfer. Set up 7 points of diluted standard such as 20ng/mL, 10ng/mL, 5ng/mL, 2.5ng/mL, 1.25ng/mL,
0.625ng/mL, 0.312ng/mL, and the last EP tubes with Standard Diluent is the blank as 0ng/mL.
3. Assay Diluent A and Assay Diluent B - Dilute 6mL of Assay Diluent A or B Concentrate（2×） with 6mL of
deionized or distilled water to prepare 12 mL of Assay Diluent A or B. （In fact, more than 6mL Assay
Diluent A and Assay Diluent B are contained in the bottles. Therefore, in every test, please precisely
pipette required amount of Diluent and make double dilution in a new container. The prepared
working dilution can't be frozen.）
Tube 1 2 3 4 5 6 7 8 9
ng/mL 200 20 10 5 2.5 1.25 0.625 0.312 0
4. Detection Reagent A and Detection Reagent B - Briefly spin or centrifuge the stock Detection A and
Detection B before use. Dilute to the working concentration with working Assay Diluent A or B, respectively
（1:100）.
5. Wash Solution - Dilute 20mL of Wash Solution concentrate （30×） with 580mL of deionized or distilled water
to prepare 600 mL of Wash Solution （1×）.
6. TMB substrate - Aspirate the needed dosage of the solution with sterilized tips and do not dump the residual
solution into the vial again.
Note:
1. Making serial dilution in the wells directly is not permitted.
2. Prepare standard within 15 minutes before assay. Please do not dissolve the reagents at 37oC directly.
3. Please carefully reconstitute Standards or working Detection Reagent A and B according to the instruction,
and avoid foaming and mix gently until the crystals are compley dissolved. To minimize imprecision caused
by pipetting, use small volumes and ensure that pipettors are calibrated. It is recommended to suck more
than 10μL for once pipetting.
4. The reconstituted Standards, Detection Reagent A and Detection Reagent B can be used only once.
5. If crystals have formed in the Wash Solution concentrate （30×）, warm to room temperature and mix gently
until the crystals are compley dissolved.
6. Contaminated water or container for reagent preparation will influence the detection result.
[ SAMPLE PREPARATION ]
1. Uscn, Inc. is only responsible for the kit itself, but not for the samples consumed during the assay. The user
should calculate the possible amount of the samples used in the whole test. Please reserve sufficient
samples in advance.
2. Please predict the concentration before assaying. If values for these are not within the range of the standard
curve, users must determine the optimal sample dilutions for their particular experiments.
3. If the samples are not indicated in the manual, a preliminary experiment to determine the validity of the kit is
necessary.
4. Tissue or cell extraction samples prepared by chemical lysis buffer may cause unexpected ELISA results due
to the impacts from certain chemicals.
5. Due to the possibility of mismatching between antigen from other origin and antibody used in our kits （e.g.,
antibody targets conformational epitope rather than linear epitope）, some native or recombinant proteins
from other manufacturers may not be recognized by our products.
6. Influenced by the factors including cell viability, cell number or sampling time, samples from cell culture
supernatant may not be detected by the kit.
7. Fresh samples without long time storage is recommended for the test. Otherwise, protein degradation and
denaturalization may occur in those samples and finally lead to wrong results.
[ ASSAY PROCEDURE ]
1. Determine wells for diluted standard, blank and sample. Prepare 7 wells for standard, 1 well for blank.
Add 100μL each of dilutions of standard （read Reagent Preparation）, blank and samples into the appropriate
wells. Cover with the Plate sealer. Incubate for 2 hours at 37oC.
2. Remove the liquid of each well, don’t wash.
3. Add 100μL of Detection Reagent A working solution to each well. Incubate for 1 hour at 37oC after covering
it with the Plate sealer.
4. Aspirate the solution and wash with 350μL of 1× Wash Solution to each well using a squirt bottle,
multi-channel pipette, manifold dispenser or autowasher, and let it sit for 1~2 minutes. Remove the remaining
liquid from all wells compley by snapping the plate onto absorbent paper. Totally wash 3 times. After the
last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and blot it against
absorbent paper.
5. Add 100μL of Detection Reagent B working solution to each well. Incubate for 30 minutes at 37oC after
covering it with the Plate sealer.
6. Repeat the aspiration/wash process for total 5 times as conducted in step 4.
7. Add 90μL of Substrate Solution to each well. Cover with a new Plate sealer. Incubate for 15 - 25 minutes at
37oC （Don't exceed 30 minutes）. Protect from light. The liquid will turn blue by the addition of Substrate
Solution.
8. Add 50μL of Stop Solution to each well. The liquid will turn yellow by the addition of Stop solution. Mix the
liquid by tapping the side of the plate. If color change does not appear uniform, gently tap the plate to ensure
thorough mixing.
9. Remove any drop of water and fingerprint on the bottom of the plate and confirm there is no bubble on the
surface of the liquid. Then, run the microplate reader and conduct measurement at 450nm immediay.
Note:
1. Assay preparation: Keep appropriate numbers of wells for 1 experiment and remove extra wells from
microplate. Rest wells should be resealed and stored at -20oC.
2. Samples or reagents addition

Please use the freshly prepared Standard. Please carefully add samples
to wells and mix gently to avoid foaming. Do not touch the well wall. For each step in the procedure, total
dispensing time for addition of reagents or samples to the assay plate should not exceed 10 minutes. This
will ensure equal elapsed time for each pipetting step, without interruption. Duplication of all standards and
specimens, although not required, is recommended. To avoid cross-contamination, change pipette tips
between additions of standards, samples, and reagents. Also, use separated reservoirs for each reagent.
3. Incubation: To ensure accurate results, proper adhesion of plate sealers during incubation steps is
necessary. Do not allow wells to sit uncovered for extended periods between incubation steps. Once
reagents are added to the well strips, DO NOT let the strips DRY at any time during the assay. Incubation
time and temperature must be controlled.
4. Washing: The wash procedure is critical. Complete removal of liquid at each step is essential for good
performance. After the last wash, remove any remaining Wash Solution by aspirating or decanting and
remove any drop of water and fingerprint on the bottom of the plate. Insufficient washing will result in poor
precision and false elevated absorbance reading.
5. Controlling of reaction time: Observe the change of color after adding TMB Substrate （e.g. observation
once every 10 minutes）, if the color is too deep, add Stop Solution in advance to avoid excessively strong
reaction which will result in inaccurate absorbance reading.
6. TMB Substrate is easily contaminated. Please protect it from light.
7. The environment humidity which is less than 60% might have some effects on the final performance,
therefore, a humidifier is recommended to be used at that condition.
[ TEST PRINCIPLE ]
The microtiter plate provided in this kit has been pre-coated with an antibody specific to uPAR. Standards or
samples are then added to the appropriate microtiter plate wells with a biotin-conjugated antibody preparation
specific for uPAR. Next, Avidin conjugated to Horseradish Peroxidase （HRP） is added to each microplate well
and incubated. After TMB substrate solution is added, only those wells that contain uPAR, biotin-conjugated
antibody and enzyme-conjugated Avidin will exhibit a change in color. The enzyme-substrate reaction is
terminated by the addition of sulphuric acid solution and the color change is measured spectrophotometrically at
a wavelength of 450nm ± 10nm. The concentration of uPAR in the samples is then determined by comparing the
O.D. of the samples to the standard curve.
[ CALCULATION OF RESULTS ]
Average the duplicate readings for each standard, control, and samples and subtract the average zero standard
optical density. Create a standard curve on log-log graph paper, with uPAR concentration on the y-axis and
absorbance on the x-axis. Draw the best fit straight line through the standard points and it can be determined by
regression analysis. Using some plot software, for instance, curve expert 1.30, is also recommended. If samples
have been diluted, the concentration read from the standard curve must be multiplied by the dilution factor.
[ TYPICAL DATA ]
In order to make the calculation easier, we plot the O.D. value of the standard （X-axis） against the known
concentration of the standard （Y-axis）, although concentration is the independent variable and O.D. value is the
dependent variable. However, the O.D. values of the standard curve may vary according to the conditions of
assay performance （e.g. operator, pipetting technique, washing technique or temperature effects）, plotting log of
the data to establish standard curve for each test is recommended. Typical standard curve below is provided for
reference only.
Typical Standard Curve for Human uPAR ELISA.
[ DETECTION RANGE ]
0.312-20ng/mL. The standard curve concentrations used for the ELISA’s were 20ng/mL, 10ng/mL, 5ng/mL,
2.5ng/mL, 1.25ng/mL, 0.625ng/mL, 0.312ng/mL.
[ SENSITIVITY ]
The minimum detectable dose of human uPAR is typically less than 0.123ng/mL.
The sensitivity of this assay, or Lower Limit of Detection （LLD） was defined as the lowest protein concentration
that could be differentiated from zero. It was determined by adding two standard deviations to the mean optical
density value of twenty zero standard replicates and calculating the corresponding concentration.
[ SPECIFICITY ]
This assay has high sensitivity and excellent specificity for detection of human uPAR.
No significant cross-reactivity or interference between human uPAR and analogues was observed.
Note:
Limited by current skills and knowledge, it is impossible for us to complete the cross- reactivity detection between
human uPAR and all the analogues, therefore, cross reaction may still exist.
[ RECOVERY ]
Matrices listed below were spiked with certain level of recombinant human uPAR and the recovery rates were
calculated by comparing the measured value to the expected amount of uPAR in samples.
Matrix Recovery range （%） Average（%）
human serum（n=5） 91-105 97
human EDTA plasma（n=5） 95-104 98
human heparin plasma（n=5） 80-96 88
[ LINEARITY ]
The linearity of the kit was assayed by testing samples spiked with appropriate concentration of human uPAR and
their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the
expected.
Sample 1

2 1

4 1

8 1

16
human serum（n=5） 95-109% 80-91% 91-107% 81-95%
human EDTA plasma（n=5） 93-101% 76-99% 90-99% 83-93%
human heparin plasma（n=5） 78-96% 94-107% 101-106% 89-98%
[ PRECISION ]
Intra-assay Precision （Precision within an assay）: 3 samples with low, middle and high level human uPAR were
tested 20 times on one plate, respectively.
Inter-assay Precision （Precision between assays）: 3 samples with low, middle and high level human uPAR were
tested on 3 different plates, 8 replicates in each plate.
CV（%） = SD/meanX100
Intra-Assay: CV<10%
Inter-Assay: CV<12%
[ STABILITY ]
The stability of ELISA kit is determined by the loss rate of activity. The loss rate of this kit is less than 5% within
the expiration date under appropriate storage condition.
The loss rate was determined by accelerated thermal degradation test. Keep the kit at 37oC for 3 days, and
compare O.D.values of the kit kept at 37oC with that of at recommended temperature. （referring from China
Biological Products Standard, which was calculated by the Arrhenius equation. For ELISA kit, 1 day storage at
37oC can be considered as 2 months at 4oC, which means 3 days at 37oC equaling 6 months at 4oC）.
Note:
To minimize extra influence on the performance, operation procedures and lab conditions, especially room
temperature, air humidity, incubator temperature should be strictly controlled. It is also strongly suggested that the
whole assay is performed by the same operator from the beginning to the end.
[ ASSAY PROCEDURE SUMMARY ]
1. Prepare all reagents, samples and standards;
2. Add 100μL standard or sample to each well. Incubate 2 hours at 37oC;
3. Add 100μL prepared Detection Reagent A. Incubate 1 hour at 37oC;
4. Aspirate and wash 3 times;
5. Add 100μL prepared Detection Reagent B. Incubate 30 minutes at 37oC;
6. Aspirate and wash 5 times;
7. Add 90μL Substrate Solution. Incubate 15-25 minutes at 37oC;
8. Add 50μL Stop Solution. Read at 450nm immediay.
[ IMPORTANT NOTE ]
1. Limited by the current condition and scientific technology, we can't compley conduct the comprehensive
identification and analysis on the raw material provided by suppliers. So there might be some qualitative and
technical risks to use the kit.
2. The final experimental results will be closely related to validity of the products, operation skills of the end
users and the experimental environments. Please make sure that sufficient samples are available.
3. Kits from different batches may be a little different in detection range, sensitivity and color developing time.
Please perform the experiment exactly according to the instruction attached in kit while electronic ones from
our website （www.uscnk.us; www.uscnk.cn; www.uscnk.com） is only for information.
4. Do not mix or substitute reagents from one kit lot to another. Use only the reagents supplied by manufacturer.
5. Protect all reagents from strong light during storage and incubation. All the bottle caps of reagents should be
covered tightly to prevent the evaporation and contamination of microorganism.
6. There may be some foggy substance in the wells when the plate is opened at the first time. It will not have
any effect on the final assay results. Do not remove microtiter plate from the storage bag until needed.
7. Wrong operations during the reagents preparation and loading, as well as incorrect parameter setting for the
plate reader may lead to incorrect results. A microplate plate reader with a bandwidth of 10nm or less and an
optical density range of 0-3 O.D. or greater at 450 ± 10nm wavelength is acceptable for use in absorbance
measurement. Please read the instruction carefully and adjust the instrument prior to the experiment. For
more information, please refer to the operation video （http://www.uscnk.com/homepage/operate-elisa.htm）.
8. Even the same operator might get different results in two separate experiments. In order to get better
reproducible results, the operation of every step in the assay should be controlled. Furthermore, a
preliminary experiment before assay for each batch is recommended.
9. Each kit has been strictly passed Q.C test. However, results from end users might be inconsistent with our
in-house data due to some unexpected transportation conditions or different lab equipments. Intra-assay
variance among kits from different batches might arise from above factors, too.
10. Kits from different manufacturers with the same item might produce different results, since we haven’t
compared our products with other manufacturers.
11. Valid period: six months.
12. The instruction manual also suits for the kit of 48T, but all reagents of 48T kit are reduced by half.
[ PRECAUTION ]
The Stop Solution suggested for use with this kit is an acid solution. Wear eye, hand, face, and clothing protection
when using this material.