EasyStep Human NGAL(Neutrophil Gelatinase
Associated Lipocalin) ELISA Kit
Cat: ELK038ES
For research use only. Not intended for diagnostic use.

Sensitivity: 5.6 pg/mL
Detection Range: 15.63-1000 pg/mL
Specificity: This assay has high sensitivity and excellent specificity for detection of Human NGAL.
No significant cross-reactivity or interference between Human NGAL and analogues was observed.
Please refer to the outer packaging label of the kit for the specific shelf life.

KIT Components
Reagents

Quantity

Storage Condition

48T

96T

Pre-Coated Microplate

6 strips x 8 wells

12 strips x 8 wells

4°C/-20°C

Standard Microplate2 strips x 8 wells

2 strips x 8 wells

4°C/-20°C

HRP Conjugate Antibody (100×)

35 μL

70 μL

4°C/-20°C(store in dark)Standard/Sample Diluent Buffer

12 mL

24 mL

4°C/-20°C

HRP Conjugate Diluent

5 mL

10 mL

4°C/-20°C

Wash Buffer (25×)

12 mL

24 mL

4°C/-20°C

TMB Substrate Solution

7 mL

12 mL

4°C/-20°C(store in dark)

Stop Reagent

4 mL

7 mL

4°C/-20°C

Plate Covers

1 piece2 pieces

4°C/-20°C

1

1

RT

Instruction Manual

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Special Explanation
1. *If the kit is opened, store Standard Microplate at -20°C, the rest reagents at 4°C. If the kit is not
used up in 1 week, please store Standard Microplate,Pre-Coated Microplate and HRP Conjugate
Antibody at -20°C, the rest reagents at 4°C, please used up within 6 months.
*If the kit is not opened, store the whole kit: 4°C(short time storage, valid for 6 months); -20°C(long-term storage, valid for 1 year). Avoid repeated freeze-thaw cycles.
2. Do not use the kit beyond the expiration date.
3. If the whole kit is stored at -20°C, place the kit at 4°C the day before the experiment.
4. After opening the package, please check that all components are complete.
5. 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 and Microplate oscillator.
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 andcorrectly.
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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.

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 Human NGAL, and the Human NGAL
standard plate wells that pre-coated using protein-related techniques are provided separately.
Standard/Sample Diluent Buffer or samples are added to the appropriate microtiter plate wells ,thenadded a HRP-conjugated antibody specific to Human NGAL . After TMB substrate solution is added, only
those wells that contain Human NGAL and HRP-conjugated antibody 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 Human
NGAL in the samples is then determined by comparing the OD of the samples to the standard curve.

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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 repeatedfreeze-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 aglass 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. For protein detection,
you can purchase our product: BC016, BCA Protein concentration determination kit.
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, andcollect them by centrifugation at 1000 × g for 5 minutes (suspension cells can be collected bycentrifugation 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.
4. Centrifuge at 1500 × g for 10 minutes at 2-8°C to remove cellular debris. Assay immediately or store
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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 washedthree 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 andstore samples at ≤-20°C. Avoid repeated freeze-thaw cycles.

Sample Dilution Proposal
Normal Human serum and plasma samples are recommended for 1:500 testing.

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 repeatedfreeze-thaw cycles.
2. The sample should be clear and transparent, and the suspended matter should be removed bycentrifugation.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.

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Summary
1. After the kit is equilibrated at room temperature, add 50μL
Standard/Sample Diluent Buffer to each Standard well ， and add 50 μL
sample to the sample well, Immediately add 50 μL 1× HRP Conjugate
Antibody Working Solution to each well，Incubate at 37°C on Microplate
oscillator for 60 min .

2. Discard the liquid in the plate, add 200 μL 1× Wash Buffer to each well,and wash the plate 5 times. After pat it dry against clean absorbent paper,
add 90 μL TMB Substrate Solution to each well, incubate at 37°C for 20
minutes in the dark.

3.

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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 theresults.

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Reagent Preparation
1. Bring all kit components and samples to room temperature (18-25°C) before use.Make sure allcomponents are dissolved and mixed well before using the kit.
1. 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.
2. Dilute the 25× Wash Buffer into 1× Wash Buffer with double-distilled Water.
3. Samples are diluted with Standard & Sample Diluent according to pre-test or sample dilution
Proposal.
4. 1× HRP Conjugate Antibody - Briefly spin or centrifuge the stock HRP Conjugate Antibody before use.
Before the experiment, the dosage required for the experiment (50 μL/ well, the actual configured
total amount must be 50-100μL greater than the calculated value) was calculated, Dilute HRP
Conjugate Antibody to the working concentration 100-fold with HRP Conjugate Diluent. The dilution
principle is to take 1 μL concentrated HRP conjugated antibody and add it to 99 μL HRP Conjugate
Diluent and mix well.
5. TMB Substrate Solution - Aspirate the needed dosage of the solution with sterilized tips and do notdump the residual solution into the vial again.

Notes
1. After receive the kit, please store the reagents according to the instructions. The plates can bedisassembled to single strips. Please use it in batches on demands.
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 ofdifferent batches cannot be mixed (except TMB, Washing Buffer and Stop Reagent).
3. The standard strips are provided separately that contains white solid standard.Re-seal the unused
standard slats and place them at -20°C for use as soon as possible.Place the standard slats upwards
as far as possible. When opening the cover, check whether there is a solid standard on the plug. If so,
the solid standard needs to be moved into the corresponding well with a pipette.
4. If the Standard/Sample Diluent Buffer is added to the standard well ,but the white solid standard on
the wall is not dissolved , it is necessary to flush the diluent in the appropriate well with a pipette to
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completely dissolve the standard product.
5. HRP Conjugate Antibody is small in volume and may be scattered in various parts of the tube duringtransportation. Please centrifuge at 1000 × g for 1 minute before use. Then, carefully pipette 4-5
times to mix the Solution. Please configure the HRP Conjugate Antibody Working Solution according
to the required amount, and use the corresponding Dilution Solution, cannot be mixed used.
6. When incubating protein and HRP conjugate antibody, it is necessary to use a micro-plate oscillator
to oscillate the Microplate. If the plate is not oscillated, the reaction will be inadequate, and the OD
value will decrease overall. The amplitude should not exceed half the height of the well, Too much
oscillation would causes the background to rise.
7. 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 notexceed 40°C), gently Mix until crystals are completely dissolved.
8. Firstly, add the Standard/Sample Diluent Buffer to the required standard wells to dissolve thestandard , and then add sample to the sample wells . The sample addition needs to be rapid. Eachsample 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.
9. 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 toaffect the microplate reader reading.
10. 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 directcontact with hands.
11. Bacterial or fungal contamination of either samples or reagents or cross-contamination, between
reagents may cause erroneous results.

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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 theStandard curve, users must determine the optimal sample dilutions for their particular experiments.

Assay Procedure
1. Before the experiment starts, all reagents should be balanced to room temperature, and all reagentsshould be prepared in advance. When diluting the reagent or sample, it is necessary to mix, and try
to avoid foaming when mixing. If the sample concentration is too high, dilute it with a sample
diluent to make the sample conform to the detection range of the kit.
2. Place the labeled standard strip in the frame of the Microplate, add 50μL Standard/Sample Diluent
Buffer to each standard well, add 50μL sample to the sample wells (if the sample needs to be diluted,
please refer to the sample dilution suggestion), pay attention to no bubbles, add the sample to thebottom of the Microplate well when adding the sample, do not touch the wall of the well. Then,each well was immediately added with 50μL HRP conjugate antibody working solution(Note: The
tips don’t touch the liquid in the wells when adding HRP conjugate antibody working solution),
Cover the microplate with Plate Cover and oscillate the Microplate with the oscillator at 500 RPM(other horizontal oscillators should adjust their own speed to ensure that the solution per well did
not exceed half height of the wells and could be mixed), and incubated at 37°C for 60 minutes
3. Discard the liquid in the wells and wash the plate 5 times. Wash each well with 200μL of washingsolution, soak for 1-2 minutes each time, and shake off the liquid in the plate (or wash the plate witha plate washer). After the last wash, pat the plate dry on absorbent paper.
4. 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.
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5. 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 asthat of the TMB Substrate Solution.
6. Wipe off any drop of water and fingerprint on the bottom of the plate and confirm there is nobubble on the surface of the liquid. Then, run the microplate reader and conduct measurement at
450 nm immediately.

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Calculation of Results
Average the duplicate readings for each Standard, Control, and Samples and subtract the averagezero Standard optical density. Construct a Standard curve with the Human NGAL 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 (pg/mL)
1000
500
250
125
62.5
31.25
15.63
0

OD
2.153
1.515
1.106
0.928
0.563
0.426
0.156
0.108

Corrected OD
2.045
1.407
0.998
0.82
0.455
0.318
0.048
0.000

Note: this graph is for reference only

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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 Human NGAL and the recovery rates
were calculated by comparing the measured value to the expected amount of Human NGAL in samples.
MatrixRecovery range

Average

Serum (n = 5)

94-105%98%

EDTA plasma (n = 5)

94-107%100%

Heparin plasma (n = 5)

82-95%

88%

Linearity
The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human
NGAL 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)

90-98%

85-92%

83-96%

93-102%EDTA plasma (n = 5)

85-94%

87-96%

82-93%

89-103%

Heparin plasma (n = 5)85-92%

83-101%

87-98%

79-92%

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Sample Value
This kit is applied to detect 8 of Serum samples of healthy Human.
Sample Type

Sample Number

Range

Mean

Positive Rate

Serum

8

127.5-197 ng/ml

155 ng/mL

100%

Note: This sample range is not physiological. The concentration range of samples varies depending on
species, sample preparation, and testing personnel and equipment. The above data is for reference only.

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 usedin 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.

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Analysis of Common Problems and Causes of ELISA ExperimentHigh background/non-specific staining
Description of
resultsAftertermination, thewhole plate
results show auniform yellow
or light color; or
the Standard
curve is linear
but thebackground is
too high

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Possible reason

Recommendations and precautions

The yellowing of the whole plate
may be caused by wrong addition ofother reagents

Check the components and lot numbers of thereagents before the experiment, and confirm
that all components belong to thecorresponding kit. Reagents from different kits
or different lot numbers cannot be mixed.

ELISA plate was not washedsufficiently

Make sure that the same amount of Washing
Solution is added to each microwell during thewashing process. After washing, press the
ELISA plate firmly on the absorbent paper toremove the residual buffer.

Incubation time too long

Please strictly follow the steps of the manual

Streptavidin-HRP contaminates the
tip and TMB container or positive
control contaminates the Pre-coatedMicroplate

When absorbing different reagents, the tips
should be replaced. When configuring different
reagent components, different storage vessels
should be used. Please use a pipette during
operation.

Streptavidin-HRP concentration too
high

Check whether the concentration calculation iscorrect or use after further dilution.

Substrate exposure orcontamination prior to use

Store in the dark at all times before adding
substrate.

Color development time is too long

Please strictly follow the steps of the manual.

The wrong filter was used when theabsorbance value was read

When TMB is used as the substrate, theabsorbance should be read at 450 nm.

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NO color plate
Description of results

After the color
development step, allwells of the ELISA plate
are colorless; the positive
control is not obvious

Possible reason

Recommendations and precautions

Mixed use of component
reagents

Please read labels clearly when
preparing or using

In the process of plate washingand sample addition, the enzyme
marker is contaminated andinactivated, and loses its ability tocatalyze the color developing
agent

Confirm that the container holding the
ELISA plate does not contain enzyme
inhibitors (such as NaN3, etc.), andconfirm that the container for preparing
the Wash Solution has been 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
preservative, which inhibits thereaction of the enzyme

Samples cannot use NaN3

The sample to be tested may notcontain strong positive samples,so the result may be normal

In case of doubt, please test again.

The visual result isnormal, but the reading
value of the microplate
reader is low

Wrong filter used for absorbancereading

When TMB is used as the substrate, theabsorbance should be read at 450 nm.

The color of all the plate
is light

No microplate oscillator is used

A microplate oscillator is required

The Standard is normal,
the color of the sample islight

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Description of
results

All wells,
includingStandard andSamples, arelighter in color

Possible reason

Recommendations and precautions

Insufficient incubation time

Timer accurate timing

Insufficient color reaction

Usually 15 - 30 minutes

The number of washings
increases, and the dilution ratioof the concentrated lotion does
not meet the requirements

Reduce the impact of washing, dilute theconcentrated lotion and washing time according to
the manual, and accurately record the washingtimes and dosage.

Distilled water quality problem

The prepared lotion must be tested to see if thepH value is neutral.

In the process of plate washingand sample addition, the enzyme
marker is contaminated andinactivated, and loses its ability tocatalyze the color developing
agent.

Confirm that the container holding the ELISA plate
does not contain enzyme inhibitors (such as NaN3,etc.), confirm that the container for preparing theWashing Solution has been washed, and confirm
that the purified water for preparing the Washing
Solution meets the requirements and is notcontaminated.

The kit has expired or been
improperly stored

Please use it within the expiration and store it inaccordance with the storage conditions
recommended in the manual to avoid
contamination.

Reagents and samples are notequilibrated before use

All reagents and samples should be equilibrated atroom temperature for about 30 minutes.

Insufficient suction volume of thepipette, too fast discharge ofpipetting suction, too much liquidhanging on the inner wall of the
tip or the inner wall is not clean.

To calibrate the pipette, the tips should bematched, each time the tips should fit tightly, thepipetting should not be too fast, and the discharge
should be complete. The inner wall of the tips
should be clean, and it is best to use it once.

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Description of
results

Possible reason

Recommendations and precautions

Incubation temperature
constant temperatureeffect is not good

Keep the temperature constant to avoid the local
temperature being too high or too low

When adding liquid, toomuch remains on the
medial wall of wells

When adding liquid, the tip should try to add liquidalong the bottom of the medial wall of wells without
touching the bottom of the hole.

Reuse of consumables

The tips should be replaced when different reagentsare drawn, and different storage vessels should be
used when configuring different reagent components.

The bottom of the
microwell is scratched or
there is dirt

Be careful when operating, be careful not to touch thebottom and wipe the bottom of the microplate toremove dirt or fingerprints.

Poor
repeatability

Technical repetition of the same sample for 3 times,
including more than 2 approximate values.

The color ofplate is chaotic
and irregular

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Cross-contamination
during sample addition

Try to avoid cross-contamination when adding samples

Cross-contaminationfrom manual plate
washing

When washing the plates by hand, the first 3 injectionsof the lotion should be discarded immediately, and thesoaking time should be set for the next few times toreduce cross-contamination.

Cross-contamination
when clapping

Use a suitable absorbent paper towel when clapping
the plate, do not pat irrelevant substances into the wellof the plate, and try not to pat in the same position to
avoid cross-contamination.

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Descriptionof results

The color ofplate ischaotic andirregular

Possible reason

Recommendations and precautions

The liquid filling head of theplate washer is blocked, resulting
in unsatisfactory liquid additionor large residual amount of liquidsuction, resulting in the color ofplate is chaotic and irregular

Unblock the liquid addition head, so that eachwell is filled with washing liquid when washing
the plate and the residual amount should besmall when aspirating liquid.

Incomplete centrifugation of thesample, resulting in coagulationin the reaction well orinterference of sediment orresidual cellular components

Serum plasma should be fully centrifuged at
3000 rpm for more than 6 minutes

The sample is stored for too longtime, resulting in contamination.

Samples should be kept fresh or stored at low
temperature to prevent contamination

Incorrect preparation of Washing
Solution or direct misuse ofconcentrated Washing Solution

Please configure according to the manual

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