Human IFNg(Interferon Gamma) ELISA Kit
Cat: ELK1036
For research use only. Not intended for diagnostic use.

Sensitivity: 5.9 pg/mL
Detection Range: 15.63-1000 pg/mL
Specificity: This assay has high sensitivity and excellent specificity for detection of Human IFNg. No
significant cross-reactivity or interference between Human IFNg and analogues was observed.
Please refer to the outer packaging label of the kit for the specific shelf life.

KIT Components
Reagents
Pre-Coated Microplate

Quantity
48T

96T

6 strips x 8 wells

Storage Condition

12 strips x 8 wells

4°C/-20°C

Standard (Lyophilized)

1 vial2 vials

4°C/-20°C

Biotinylated Antibody (100×)60 μL

120 μL

4°C/-20°C

Streptavidin-HRP (100×)60 μL

120 μL

4°C/-20°C

Standard/Sample Diluent
Buffer

10 mL

20 mL

4°C/-20°C

Biotinylated Antibody Diluent

6 mL

12 mL

4°C/-20°C

HRP Diluent

6 mL

12 mL

4°C/-20°C

Wash Buffer (25×)

10 mL

20 mL

4°C/-20°C

TMB Substrate Solution

6 mL

10 mL

4°C/-20°C (store in dark)

Stop Reagent

3 mL

6 mL

4°C/-20°C

Plate Covers

1 piece2 piecesRT

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Special Explanation
1. *If the kit is opened, Store the whole kit at 4°C. If the kit is not used up in 1 week. Store the
Pre-Coated Microplate, Standard ,Biotinylated Antibody and Streptavidin-HRP 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.
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 IFNg. Standards or samples
are added to the appropriate microtiter plate wells then with a biotin-conjugated antibody specific to
Human IFNg. Next, Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate welland incubated. After TMB substrate solution is added, only those wells that contain Human IFNg,
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 Human
IFNg 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.

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. 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 100 μL of Standard Working Buffer(gradually diluted according to the instructions) or 100 μL of sample to each well, andincubate at 37°C for 80 minutes.

2. Discard the liquid in the plate, add 200 μL 1× Wash Buffer to each well, and wash theplate 3 times. After pat it dry against clean absorbent paper, 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 theplate 3 times. After pat it dry against clean absorbent paper, add 100 μL 1×
Streptavidin-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 theplate 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.

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.

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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.
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 theStandard with 1.0 mL of Standard Diluent Buffer, kept for 10 minutes at room temperature, shakegently (not to foam). The concentration of the Standard in the stock solution is 1000 pg/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 1000 pg/mL, 500 pg/mL, 250 pg/mL, 125 pg/mL, 62.5 pg/mL, 31.25 pg/mL, 15.63
pg/mL, and the last EP tubes with Standard Diluent is the Blank as 0 pg/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 eachdilution. Note: the last tube is regarded as a Blank and do not pipette solution into it from theformer tube.

Stock
Standard
pg/mL

1000

500

250

125

62.5

31.25

15.63

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.
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6. 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.

Lyophilized Standards, Biotinylated Antibody, and Streptavidin-HRP are small in volume and may bescattered 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 theStandard, 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 notexceed 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 additionneeds 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 toaffect the microplate reader reading.

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,

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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.
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 theStandard 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 toeach well and let it sit for 1-2 minutes. Remove the remaining liquid from all wells completely bysnapping 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.
Notes: (a) When adding Washing Solution, the pipette tip should not touch the wall of the wells to avoid contamination.
(b) Pay attention to pouring the washing liquid directly to ensure that the washing liquid does notcontaminate other wells.
3. Add 100 μL of Biotinylated Antibody Working Solution to each well, cover the wells with the Plate
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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 asthat of the TMB Substrate Solution.
9. 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 IFNg 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
2.128
1.482
0.896
0.543
0.306
0.251
0.222
0.13

Corrected OD
1.998
1.352
0.766
0.413
0.176
0.121
0.092
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 IFNg and the recovery rates
were calculated by comparing the measured value to the expected amount of Human IFNg in samples.
MatrixRecovery range

Average

Serum (n = 5)

96-107%101%

EDTA plasma (n = 5)

87-99%

92%

Heparin plasma (n = 5)

84-96%

90%

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

86-93%

91-103%87-98%

95-101%EDTA plasma (n = 5)

89-99%

83-95%

98-104%96-106%

Heparin plasma (n = 5)

85-100%84-98%

95-101%

88-102%

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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.

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.

Biotinylated Antibody or
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
The Standard is normal,
the color of the sample islight

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

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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.

Wrong filter used for absorbancereading

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

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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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