Lablisa® Human TIMP1(Tissue

Inhibitors OfMetalloproteinase 1)
ELISA Kit
Cat: LAB1259
96 Tests
For research use only. Not intended for diagnostic use.

Sensitivity: 0.059 ng/mL

Detection Range: 0.16-10 ng/mL
Specificity: This assay has high sensitivity and excellent specificity for detection of Human TIMP1.
No significant cross-reactivity or interference between Human TIMP1 and analogues was observed.

Please refer to the outer packaging label of the kit for the specific shelf life.

KIT Components & Storage

Store the kit at 4°C for 1 week. If the kit is not used up in 1 week, store the items separately according to
the following conditions after the kit is received.

Quantity
Reagents Storage Condition
48T 96T
6 strips x 8 wells 12 strips x 8 wells -20°C (6 months)
Pre-Coated Microplate
Standard (Lyophilized) 1 vial 2 vials -20°C (6 months)
Biotinylated Antibody (100×) 60 μL 120 μL -20°C (6 months)
Streptavidin-HRP (100×) 60 μL 120 μL -20°C (6 months)
Standard/Sample Diluent Buffer 10 mL 20 mL 4°C
Biotinylated Antibody Diluent 6 mL 12 mL 4°C
HRP Diluent 6 mL 12 mL 4°C
Wash Buffer (25×) 10 mL 20 mL 4°C
TMB Substrate Solution 6 mL 10 mL 4°C (store in dark)
3 mL 6 mL
Stop Reagent 4°C
Plate Covers 1 piece 2 pieces 4°C

www.labrecon.in 1 support@labrecon.in
Special Explanation

1. Please store kit at 4°C if used up in 1 week. If used for more than 1 week, store the Pre-Coated
Microplate, Standard, Biotinylated Antibody and Streptavidin-HRP at -20°C and all other reagents at
4°C according to the temperature indicated on the label. Avoid repeated freeze-thaw cycles.
2. Do not use the kit beyond the expiration date.
3. After opening the package, please check that all components are complete.
4. The cap must be tightened to prevent evaporation and microbial contamination. The reagents
volume is slightly more than the volume marked on labels, please use accurate measuring
equipment and do not pour directly into the vial.
All kit components have been formulated and quality control tested to function successfully. Do not mix
or substitute reagents or materials from other kits, detection effect of the kit will not be guaranteed if
utilized separately or substituted.

Materials Required, Not Supplied

1. Microplate reader capable of measuring absorbance at 450 ± 10 nm.

2. High-speed centrifuge.
3. Electro-heating standing-temperature cultivator.
4. Absorbent paper.
5. Double distilled water or deionized water.
6. Single or multi-channel pipettes with high precision and disposable tips.
7. Precision pipettes to deliver 2 μL to 1 mL volumes.

Safety Notes

1. This kit is only used for lab research and development and should not be used for human or animals.
2. Reagents should be regarded as hazardous substances and should be handled carefully and
correctly.
3. Gloves, lab coats, and goggles should always be worn to avoid skin and eyes coming into contact
with Stop Solution and TMB. In case of contact, wash thoroughly with water.

www.labrecon.in 2 support@labrecon.in
Test Principle

The test principle applied in this kit is Sandwich enzyme immunoassay. The microtiter plate
provided in this kit has been pre-coated with an antibody specific to Tissue Inhibitors Of
Metalloproteinase 1(TIMP1). Standards or samples are added to the appropriate microtiter plate wells
then with a biotin-conjugated antibody specific to Tissue Inhibitors Of Metalloproteinase 1(TIMP1). 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 Tissue Inhibitors Of Metalloproteinase
1(TIMP1), 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

Tissue Inhibitors Of Metalloproteinase 1(TIMP1) in the samples is then determined by comparing the OD
of the samples to the standard curve.

www.labrecon.in 3 support@labrecon.in
Sample Collection and Storage

Serum - Samples should be collected into a serum separator tube. After clotting for 2 hours at room
temperature or overnight at 4°C, and then centrifuging at 1000 × g for 20 minutes. Assay freshly
prepared serum immediately or store samples in aliquot at -20°C or -80°C for later use. Avoid repeated
freeze-thaw cycles.
Plasma - Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples at 1000 × g and
2-8°C for 15 minutes within 30 minutes of collection. Remove plasma and assay immediately or store
samples in aliquot at -20°C or -80°C for later use. Avoid repeated freeze-thaw cycles.
Tissue homogenates - The preparation of tissue homogenates will vary depending upon tissue type.
1. Rinse the tissues in pre-cooled PBS to completely remove excess blood, and weigh them before
homogenization.
2. Mince the tissues to small pieces and homogenized them in fresh lysis buffer (different lysis buffer
needs to be chosen based on subcellular location of the target protein) (PBS can be used as the lysis
buffer of most tissues) (w:v = 1:9, e.g. 900 µL lysis buffer is added in 100 mg tissue sample) with a
glass homogenizer on ice (micro tissue grinders, too).
3. Ultrasound the obtained suspension with an ultrasonic cell disrupter until the solution is clear.
4. Then, centrifuge the homogenates for 5 minutes at 10000 × g and collect the supernatant and assay
immediately or store in aliquots at ≤ -20°C.
*Note: Tissue homogenates are recommended to be tested for protein concentration at the same time
to obtain a more accurate concentration of the test substance per mg of protein.
Cell lysates - Cells need to be lysed before assaying according to the following directions.
1. Adherent cells should be washed by pre-cooled PBS gently, and then be detached with trypsin, and
collect them by centrifugation at 1000 × g for 5 minutes (suspension cells can be collected by
centrifugation directly).
2. Wash cells 3 times in pre-cooled PBS.
3. Then, resuspend the cells in fresh lysis buffer with concentration of 107 cells/mL. If it is necessary,
the cells could be subjected to ultrasonication until the solution is clear.

www.labrecon.in 4 support@labrecon.in
4. Centrifuge at 1500 × g for 10 minutes at 2-8°C to remove cellular debris. Assay immediately or store
in aliquots at ≤ -20°C.
Urine - Collect the first urine of the day (mid-stream) and discharge it directly into a sterile container.
Centrifuge to remove particulate matter, assay immediately or aliquot and store at ≤ -20°C. Avoid
repeated freeze-thaw cycles.
Saliva - Collect saliva using a collection device or equivalent. Centrifuge samples at 1000 × g at 2-8°C for
15 minutes. Remove particulates and assay immediately or store samples in aliquot at ≤ -20°C. Avoid
repeated freeze-thaw cycles.
Feces - Dry feces were collected as much as possible, weighing more than 50 mg. The feces were washed
three times with PBS (w:v = 1:9, e.g. 900 µL lysis buffer is added in 100 mg feces), sonicated (or mashed)
and centrifuged at 5000×g for 10 minutes, where the supernatant was collected for testing.
Cell culture supernatants and other biological fluids - Centrifuge samples at 1000 × g for 20 minutes.
Collect the supernatant and assay immediately or store samples in aliquot at -20°C or -80°C for later use.
Avoid repeated freeze-thaw cycles.
Cerebrospinal fluid (CSF) - Remove particulates by centrifugation and assay immediately or aliquot and
store samples at ≤ -20°C. Avoid repeated freeze-thaw cycles.

Notes

1. Samples to be used within 5 days may be stored at 4°C, otherwise samples must be stored at -20°C
(≤ 1 month) or -80°C (≤ 2 months) to avoid loss of bioactivity and contamination. Avoid repeated
freeze-thaw cycles.
2. Sample hemolysis will influence the result, so it should not be used.
3. When performing the assay, bring samples to room temperature.
4. If the concentration of the test material in your sample is higher than that of the Standard product,
please make the appropriate multiple dilutions according to the actual situation (it is recommended
to do preliminary experiment to determine the dilution ratio.

www.labrecon.in 5 support@labrecon.in
Summary

1. After the kit is equilibrated at room temperature, add 100 μL of Standard Working Buffer
(gradually diluted according to the instructions) or 100 μL of sample to each well, and
incubate at 37°C for 80 minutes.

2. Discard the liquid in the plate, add 200 μL 1 ×Wash Buffer to each well, and wash the
plate 3 times. After spin-drying, add 100 μL Biotinylated Antibody Working Solution (1×) to
each well, incubate at 37°C for 50 minutes.

3. Discard the liquid in the plate, add 200 μL 1 ×Wash Buffer to each well, and wash the
plate 3 times. After drying, add 100 μL 1×Streptavdin-HRP Working Solution to each well,
incubate at 37°C for 50 minutes.

4. Discard the liquid in the plate, add 200 μL 1×Wash Buffer to each well, and wash the
plate 5 times. After spin-drying, add 90 μL TMB Substrate Solution to each well, incubate at
37°C for 20 minutes in the dark.

5. Add 50 μL Stop Solution to each well, shake plate on a plate shaker for 1 minute to mix.
Record the OD at 450 nm immediately, calculation of the results.

www.labrecon.in 6 support@labrecon.in
Reagent Preparation

1. Bring all kit components and samples to room temperature (18-25°C) before use.
2. If the kit will not be used up in 1 time, please only take out strips and reagents for present
experiment, and save the remaining strips and reagents as specified.

3. Dilute the 25×Wash Buffer into 1×Wash Buffer with double-distilled Water.
4. Standard Working Solution - Centrifuge the Standard at 1000 × g for 1 minute. Reconstitute the
Standard with 1.0 mL of Standard Diluent Buffer, kept for 10 minutes at room temperature, shake
gently (not to foam). The concentration of the Standard in the stock solution is 10 ng/mL. Please
prepare 7 tubes containing 0.5 mL Standard Diluent Buffer and use the Diluted Standard to produce
a double dilution series according to the picture shown below. To mix each tube thoroughly before
the next transfer, pipette the solution up and down several times. Set up 7 points of Diluted
Standard such as 10 ng/mL, 5 ng/mL, 2.5 ng/mL, 1.25 ng/mL, 0.63 ng/mL, 0.32 ng/mL, 0.16 ng/mL,
and the last EP tubes with Standard Diluent is the Blank as 0 ng/mL. In order to guarantee the
experimental results validity, please use the new Standard Solution for each experiment. When
diluting the Standard from high concentration to low concentration, replace the pipette tip for each
dilution. Note: the last tube is regarded as a Blank and do not pipette solution into it from the
former tube.

Stock
Standard

ng/mL 10 5 2.5 1.25 0.63 0.32 0.16

5. 1×Biotinylated Antibody and 1×Streptavidin-HRP - Briefly spin or centrifuge the stock

Biotinylated Antibody and Streptavidin-HRP before use. Dilute them to the working concentration
100-fold with Biotinylated Antibody Diluent and HRP Diluent, respectively.

www.labrecon.in 7 support@labrecon.in
6. TMB Substrate Solution - Aspirate the needed dosage of the solution with sterilized tips and do not
dump the residual solution into the vial again.

Notes

1. After receive the kit, please store the reagents according to the instructions. The plates can be
disassembled to single strips. Please use it in batches on demands. It is recommended that the
remaining reagents are used within 1 month after the first test.
2. The test tubes, pipette tips and reagents used in the experiment are all disposable and are strictly
prohibited from being reused; otherwise the experiment results will be affected. Kit reagents of
different batches cannot be mixed (except TMB, Washing Buffer and Stop Reagent).
3. Lyophilized Standards, Biotinylated Antibody, and Streptavidin-HRP are small in volume and may be
scattered in various parts of the tube during transportation. Please centrifuge at 1000 × g for 1
minute before use. Then, carefully pipette 4-5 times to mix the Solution. Please configure the
Standard, Biotinylated Antibody and Streptavidin-HRP Working Solution according to the required
amount, and use the corresponding Dilution Solution, cannot be mixed used.
4. Bring all reagents to room temperature (18-25°C) before use. If crystals form in the concentrate (25
×), it is a normal phenomenon. Heat it to room temperature (the heating temperature should not
exceed 40°C), gently Mix until crystals are completely dissolved.
5. Prepare to dissolve Standard within 15 minutes before the test. This Standard Working Solution can
only be used once. If the dissolved Standard is not used up, please discard it. The sample addition
needs to be rapid. Each sample addition should preferably be controlled within 10 minutes. To
ensure experimental accuracy, it is recommended to test duplicate wells, and when pipetting
reagents, keep a consistent order of additions from 1 well to another, this will ensure the same
incubation time for all wells.
6. During the washing process, the residual washing liquid in the reaction well should be patted dry on
absorbent paper. Do not put the paper directly into the reaction well to absorb water. Before
reading, pay attention to remove the residual liquid and fingerprints at the bottom, so as not to
affect the microplate reader reading.

www.labrecon.in 8 support@labrecon.in
7. TMB Substrate Solution is light-sensitive, avoid prolonged exposure to light. Dispense the TMB
Substrate Solution within 15 minutes following the washing of the microtiter plate. In addition,
avoid contact between TMB Substrate Solution and metal to prevent color development. TMB is
contaminated if it turns blue color before use and should be discarded. TMB is toxic, avoid direct
contact with hands.
8. Bacterial or fungal contamination of either samples or reagents or cross-contamination, between
reagents may cause erroneous results.

Samples Preparation

1. Equilibrate all materials and prepared reagents to room temperature prior to use. Prior to use, mix
all reagents thoroughly taking care not to create any foam within the vials.
2. The user should calculate the possible amount of the samples used in the whole test. Please reserve
sufficient samples in advance.
3. 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.

Assay Procedure

1. Determine wells for Diluted Standard, Blank and Sample. Prepare 7 wells for Standard, 1 well for
Blank. Add 100 μL each of Standard Working Solution (please refer to Reagent Preparation), or 100
μL of samples into the appropriate wells. Cover with the Plate Cover. Incubate for 80 minutes at
37°C. Note: solutions should be added to the bottom of the micro ELISA plate well, avoid touching
the inside wall and causing foaming as much as possible.

2. Pour out the liquid of each well. Aspirate the solution and wash with 200 μL of 1×Wash Solution to
each well and let it sit for 1-2 minutes. Remove the remaining liquid from all wells completely 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.

www.labrecon.in 9 support@labrecon.in
 Notes: (a) When adding Washing Solution, the pipette tip should not touch the wall of the wells to a
void contamination.
(b) Pay attention to pouring the washing liquid directly to ensure that the washing liquid does not
contaminate other wells.
3. Add 100 μL of Biotinylated Antibody Working Solution to each well, cover the wells with the Plate
Cover and incubate for 50 minutes at 37°C.

4. Repeat the aspiration,wash process for total 3 times as conducted in step 2.

5. Add 100 μL of Streptavidin-HRP Working Solution to each well, cover the wells with the plate sealer
and incubate for 50 minutes at 37°C.
6. Repeat the aspiration, wash process for total 5 times as conducted in step 2.
7. Add 90 μL of TMB Substrate Solution to each well. Cover with a new Plate Cover. Incubate for 20
minutes at 37°C (Don't exceed 30 minutes) in the dark. The liquid will turn blue by the addition of
TMB Substrate Solution. Preheat the Microplate Reader for about 15 minutes before OD
measurement.
8. Add 50 μL of Stop Reagent to each well. The liquid will turn yellow by the addition of Stop Reagent.
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. The insertion order of the Stop Reagent should be the same as
that of the TMB Substrate Solution.
9. Wipe off 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
450 nm immediately.

www.labrecon.in 10 support@labrecon.in
Calculation of Results

Average the duplicate readings for each Standard, Control, and Samples and subtract the average
zero Standard optical density. Construct a Standard curve with the Human TIMP1 concentration on the
y-axis and absorbance on the x-axis, and draw a best fit curve through the points on the graph. If
samples have been diluted, the concentration read from the Standard curve must be multiplied by the
dilution factor. Using some plot software, for instance, curve expert.

Concentration (ng/mL) OD Corrected OD

10 2.058 1.99
5 1.562 1.494
2.5 1.143 1.075
1.25 0.797 0.729
0.63 0.519 0.451
0.32 0.352 0.284
0.16 0.211 0.143
0 0.068 0.000

Note: this graph is for reference only

www.labrecon.in 11 support@labrecon.in
Precision

Intra-assay Precision (Precision within an assay)：CV% < 8%

Three samples of known concentration were tested twenty times on 1 plate to assess intra-assay
precision.

Inter-assay Precision (precision between assays)：CV% < 10%

Three samples of known concentration were tested in forty separate assays to assess inter-assay
precision.

Recovery

Matrices listed below were spiked with certain level of recombinant TIMP1 and the recovery rates were
calculated by comparing the measured value to the expected amount of TIMP1 in samples.

Matrix Recovery range Average

Serum (n = 5) 86-99% 92%

EDTA plasma (n = 5) 79-93% 86%

Heparin plasma (n = 5) 90-105% 97%

Linearity

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of TIMP1
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

Serum (n = 5) 83-95% 88-102% 85-97% 80-92%

EDTA plasma (n = 5) 98-107% 93-102% 80-93% 91-100%

Heparin plasma (n = 5) 88-95% 98-107% 90-99% 88-97%

www.labrecon.in 12 support@labrecon.in
Declaration

1. The kit may not be suitable for special experimental samples where the validity of the experiment
itself is uncertain, such as gene knockout experiments.
2. Certain natural or recombinant proteins, including prokaryotic and eukaryotic recombinant proteins,
may not be detected because they do not match the detection antibody and capture antibody used
in this product.
3. This kit is not compared with similar kits from other manufacturers or products with different
methods to detect the same object, so inconsistent test results cannot be ruled out.

Analysis of Common Problems and Causes of ELISA Experiment

High background/non-specific staining

Description of
Possible reason Recommendations and precautions
results
The yellowing of the whole Check the components and lot numbers of the
plate may be caused by reagents before the experiment, and confirm
wrong addition of other that all components belong to the
reagents corresponding kit. Reagents from different kits
or different lot numbers cannot be mixed.
ELISA plate was not washed Make sure that the same amount of Washing
sufficiently Solution is added to each microwell during the
After
washing process. After washing, press the ELISA
termination, the
plate firmly on the absorbent paper to remove
whole plate
the residual buffer.
results show a
Incubation time too long Please strictly follow the steps of the manual
uniform yellow
Streptavidin-HRP When absorbing different reagents, the tips
or light color; or
contaminates the tip and should be replaced. When configuring different
the Standard
TMB container or positive reagent components, different storage vessels
curve is linear
control contaminates the should be used. Please use a pipette during
but the
Pre-coated Microplate operation.
background is
Biotinylated Antibody or Check whether the concentration calculation is
too high
Streptavidin-HRP correct or use after further dilution.
concentration too high
Substrate exposure or Store in the dark at all times before adding
contamination prior to use substrate.
Color development time is Please strictly follow the steps of the manual.
too long
The wrong filter was used When TMB is used as the substrate, the
www.labrecon.in 13 support@labrecon.in
 when the absorbance value absorbance should be read at 450 nm.
was read

NO color plate

Description of results Possible reason Recommendations and precautions

Mixed use of component Please read labels clearly when
reagents preparing or using

After the color In the process of plate washing Confirm that the container holding the
development step, all and sample addition, the ELISA plate does not contain enzyme
wells of the ELISA plate enzyme marker is inhibitors (such as NaN3, etc.), and
are colorless; the contaminated and inactivated, confirm that the container for
positive control is not and loses its ability to catalyze preparing the Wash Solution has been
obvious the color developing agent washed.

Missing a reagent or a step Review the manual in detail and

strictly follow the operating steps

Light color

Description of results Possible reason Recommendations and precautions

The sample uses NaN3 Samples cannot use NaN3
preservative, which inhibits the
The Standard is normal, reaction of the enzyme
the color of the sample The sample to be tested may In case of doubt, please test again.
is light not contain strong positive
samples, so the result may be
normal
The visual result is Wrong filter used for When TMB is used as the substrate,
normal, but the reading absorbance reading the absorbance should be read at 450
value of the microplate nm.
reader is low

www.labrecon.in 14 support@labrecon.in
 Description of
Possible reason Recommendations and precautions
results
Insufficient incubation time Timer accurate timing
Insufficient color reaction Usually 15 - 30 minutes
The number of washings Reduce the impact of washing, dilute the
increases, and the dilution ratio concentrated lotion and washing time according
of the concentrated lotion does to the manual, and accurately record the
not meet the requirements washing times and dosage.
Distilled water quality problem The prepared lotion must be tested to see if the
pH value is neutral.
In the process of plate washing Confirm that the container holding the ELISA
and sample addition, the plate does not contain enzyme inhibitors (such
enzyme marker is contaminated as NaN3, etc.), confirm that the container for
and inactivated, and loses its preparing the Washing Solution has been
All wells,
ability to catalyze the color washed, and confirm that the purified water for
including
developing agent. preparing the Washing Solution meets the
Standard and
requirements and is not contaminated.
Samples, are
The kit has expired or been Please use it within the expiration and store it in
lighter in color
improperly stored accordance with the storage conditions
recommended in the manual to avoid
contamination.
Reagents and samples are not All reagents and samples should be equilibrated
equilibrated before use at room temperature for about 30 minutes.

Insufficient suction volume of To calibrate the pipette, the tips should be

the pipette, too fast discharge matched, each time the tips should fit tightly,
of pipetting suction, too much the pipetting should not be too fast, and the
liquid hanging on the inner wall discharge should be complete. The inner wall of
of the tip or the inner wall is the tips should be clean, and it is best to use it
not clean. once.

www.labrecon.in 15 support@labrecon.in
 Description of
Possible reason Recommendations and precautions
results
Incubation temperature Keep the temperature constant to avoid the local
constant temperature temperature being too high or too low
effect is not good
When adding liquid, too When adding liquid, the tip should try to add liquid
much remains on the along the bottom of the medial wall of wells without
medial wall of wells touching the bottom of the hole.
Reuse of consumables The tips should be replaced when different reagents
Poor are drawn, and different storage vessels should be
repeatability used when configuring different reagent components.
The bottom of the Be careful when operating, be careful not to touch the
microwell is scratched or bottom and wipe the bottom of the microplate to
there is dirt remove dirt or fingerprints.
Technical repetition of the same sample for 3 times,
including more than 2 approximate values.
Cross-contamination Try to avoid cross-contamination when adding samples
during sample addition
Cross-contamination When washing the plates by hand, the first 3 injections
from manual plate of the lotion should be discarded immediately, and the
washing soaking time should be set for the next few times to
The color of
reduce cross-contamination.
plate is chaotic
Cross-contamination Use a suitable absorbent paper towel when clapping
and irregular
when clapping the plate, do not pat irrelevant substances into the well
of the plate, and try not to pat in the same position to
avoid cross-contamination.

www.labrecon.in 16 support@labrecon.in
 Description
Possible reason Recommendations and precautions
of results
The liquid filling head of the Unblock the liquid addition head, so that each
plate washer is blocked, resulting well is filled with washing liquid when washing
in unsatisfactory liquid addition the plate and the residual amount should be
or large residual amount of liquid small when aspirating liquid.
suction, resulting in the color of
plate is chaotic and irregular
The color of Incomplete centrifugation of the Serum plasma should be fully centrifuged at
plate is sample, resulting in coagulation 3000 rpm for more than 6 minutes
chaotic and in the reaction well or
irregular interference of sediment or
residual cellular components
The sample is stored for too long Samples should be kept fresh or stored at low
time, resulting in contamination. temperature to prevent contamination
Incorrect preparation of Washing Please configure according to the manual
Solution or direct misuse of
concentrated Washing Solution

STATEMENT: This manual is for your reference only; specifications are subject to the delivery manuals with the original
product.

www.labrecon.in 17 support@labrecon.in