ab181421 – Human TNF alpha SimpleStep ELISA® Kit
For the quantitative measurement of TNF alpha in human serum, plasma (heparin), plasma(EDTA), plasma (citrate), cell culture supernatant, and CSF.
For research use only - not intended for diagnostic use.
For overview, typical data and additional information please visit: www.abcam.com/ab181421
This kit is available in a 384-well plate format. This plate utilises smaller volumes of standards andsamples per well. Directions for using this format can be found on pg 9.

Storage and Stability: Store kit at 2-8ºC immediately upon receipt. Refer to list of materials
supplied for storage conditions of individual components. Observe the storage conditions forindividual prepared components in the Standard Preparation and Reagent preparation
sections.

Materials Supplied
Item

Human TNF alpha Capture Antibody 10X
Human TNF alpha Detector Antibody 10X
Human TNF alpha Lyophilized RecombinantProtein
Antibody Diluent 4BR
Sample Diluent NS
Wash Buffer PT 10X
TMB Development Solution
Stop Solution
SimpleStep Pre-Coated 96-Well Microplate
Plate Seal

Quantity
1 x 96 wells

Quantity
10 x 96 wells

Storage
Condition

600 μL
600 μL
2 Vials

10 x 600 μL
10 x 600 μL
10 x 2 vials

+4°C
+4°C
+4°C

6 mL
50 mL
20 mL
12 mL
12 mL
96 wells
1

10 x 6 mL
2 x 250 mL
200 mL
120 mL
120 mL10 x 96 wells
10

+4°C
+4°C
+4°C
+4°C
+4°C
+4°C
+4°C

Materials Required, Not Supplied
These materials are not included in the kit, but will be required to successfully utilize this assay:
Microplate reader capable of measuring absorbance at 450 or 600 nm.
Deionized water.
Multi- and single-channel pipettes.
Tubes for standard dilution.
Plate shaker for all incubation steps.
Optional: Phenylmethylsulfonyl Fluoride (PMSF) (or other protease inhibitors).

Reagent Preparation
Equilibrate all reagents to room temperature (18-25°C) prior to use. The kit contains enoughreagents for 96 wells. The sample volumes below are sufficient for 48 wells (6 x 8-well strips);adjust volumes as needed for the number of strips in your experiment.
Prepare only as much reagent as is needed on the day of the experiment. Capture anddetector antibodies have only been tested for stability in the provided 10X formulations.

1X Wash Buffer PT: Prepare 1X Wash Buffer PT by diluting Wash Buffer PT 10X with deionizedwater. To make 50 mL 1X Wash Buffer PT combine 5 mL Wash Buffer PT 10X with 45 mL deionizedwater. Mix thoroughly and gently.
Antibody Cocktail: Prepare Antibody Cocktail by diluting the capture and detector antibodies
in Antibody Diluent 4BR . To make 3 mL of the Antibody Cocktail combine 300 µL 10X Capture
Antibody and 300 µL 10X Detector Antibody with 2.4 mL Antibody Diluent 4BR . Mix thoroughly
and gently.

Standard Preparation
Always prepare a fresh set of standards for every use. Discard working standard dilutions after
use as they do not store well. The following section describes the preparation of a standard
curve
for
duplicatemeasurements(recommended).
1.

2.
3.
4.

Reconstitute the TNF alpha standard sample by adding the volume of Sample Diluent NS
indicated on the protein vial label. Hold at room temperature for 10 minutes. Mix
thoroughly and gently. This is the 10,000 pg/mL Stock Standard Solution.
Label eight tubes, Standards 1– 8.
Add 360 μL of Sample Diluent NS into tube number 1 and 150 μL of Sample Diluent NS into
numbers 2-8.
Use the Stock Standard to prepare the following dilution series. Standard #8 contains noprotein and is the Blank control:Standard #Dilution
SampleVolume to
Dilute (μL)

Volume ofDiluent (μL)

StartingConc.
(pg/mL)

Final
Conc.
(pg/mL)

1

Stock Standard

40

360

10,0001,000

2Standard#1

150

150

1,000

500

3

Standard#2

150

150

500

250

4

Standard#3

150

150

250

125

5

Standard#4

150

150

125

62.5

6

Standard#5

150

150

62.5

31.25

7

Standard#6

150

150

31.25

15.63

8

Blank Control

0

150

0

0

Sample Preparation

Assay Procedure
Typical Sample Dynamic Range
Sample Type

Range

Serum*≤1:40

Plasma – Citrate*≤1:40

Plasma – EDTA*≤1:40

Plasma – Heparin*≤1:40

PBMC Cell Culture Supernatant**≤1:80 – 1:10

Cerebrospinal Fluid*≤1:2

Equilibrate all materials and prepared reagents to room temperature prior to use.
We recommend that you assay all standards, controls and samples in duplicate.
1.
2.
3.
4.
5.
6.

*Based on spiked sample**Range can vary depending on stimulation factors
Serum Samples should be collected into a serum separator tube. After clot formation,
centrifuge samples at 2,000 x g for 10 minutes and collect serum. Dilute samples at least 1:40
into Sample Diluent NS and assay. Store un-diluted serum at -20ºC or below. Avoid repeatedfreeze-thaw cycles.
Plasma Collect plasma using citrate, EDTA or heparin. Centrifuge samples at 2,000 x g for 10
minutes. Dilute samples at least 1:40 into Sample Diluent NS and assay. Store un-diluted plasma
samples at -20ºC or below for up to 3 months. Avoid repeated freeze-thaw cycles.
Cell Culture Supernatants Centrifuge cell culture media at 2,000 x g for 10 minutes to remove
debris. Collect supernatants and assay. Or dilute samples at least 1:10 into Sample Diluent NS
and assay. Store un-diluted samples at -20°C or below. Avoid repeated freeze-thaw cycles.
Cerebrospinal Fluid (CSF) Dilute cerebrospinal fluid at least 1:2 into Sample Diluent NS andassay. Store un-diluted samples at -20°C or below. Avoid repeated freeze-thaw cycles.

7.

Prepare all reagents, working standards, and samples as directed in the previous sections.
Remove excess microplate strips from the plate frame, return them to the foil pouchcontaining the desiccant pack, reseal and return to 4ºC storage.
Add 50 µL of all sample or standard to appropriate wells.
Add 50 µL of the Antibody Cocktail to each well.
Seal the plate and incubate for 1 hour at room temperature on a plate shaker set to
400 rpm.
Wash each well with 3 x 350 µL 1X Wash Buffer PT. Wash by aspirating or decanting fromwells then dispensing 350 µL 1X Wash Buffer PT into each well. Wash Buffer PT should remain
in wells for at least 10 seconds. Complete removal of liquid at each step is essential forgood performance. After the last wash invert the plate and tap gently against clean
paper towels to remove excess liquid.
Add 100 µL of TMB Development Solution to each well and incubate for 10 minutes in thedark on a plate shaker set to 400 rpm.

Given variability in laboratory environmental conditions, optimal incubation time may vary
between 5 and 20 minutes.

8.
9.

Note: The addition of Stop Solution will change the color from blue to yellow and enhance
the signal intensity about 3X. To avoid signal saturation, proceed to the next step before
the high concentration of the standard reaches a blue color of O.D.600 equal to 1.0.
Add 100 µL of Stop Solution to each well. Shake plate on a plate shaker for 1 minute tomix. Record the OD at 450 nm. This is an endpoint reading.
Alternative to 7 – 8: Instead of the endpoint reading at 450 nm, record the developmentof TMB Substrate kinetically. Immediately after addition of TMB Development Solution
begin recording the blue color development with elapsed time in the microplate reader
prepared with the following settings:

Plate Preparation
The 96 well plate strips included with this kit are supplied ready to use. It is not necessary to rinse
the plate prior to adding reagents.
Unused plate strips should be immediately returned to the foil pouch containing the desiccantpack, resealed and stored at 4°C.
For each assay performed, a minimum of two wells must be used as the zero control.
For statistical reasons, we recommend each sample should be assayed with a minimum of two
replicates (duplicates).
Differences in well absorbance or “edge effects” have not been observed with this assay.

Mode

Kinetic

Wavelength:

600 nmTime:

up to 20 min

Interval:

20 sec - 1 min

Shaking:

Shake between readings

Note that an endpoint reading can also be recorded at the completion of the kinetic
read by adding 100 µL Stop Solution to each well and recording the OD at 450 nm.
Download our ELISA guide for technical hints, results, calculation, and troubleshooting tips:
www.abcam.com/protocols/the-complete-elisa-guide
For technical support contact information, visit: www.abcam.com/contactus

Copyright © 2023 Abcam, All Rights Reserved. All information / detail is correct at time of going to print.
Version 8 | 2023-10-10

ab181421 – Human TNF alpha SimpleStep ELISA® Kit
Additional information

CALCULATION-ASSAY SPECIFICITYThis kit is designed for the quantification of human TNF alpha.
Native signal was detected in cell culture supernatant.
Spiked protein experiments were used to validate serum, plasma (heparin), plasma (EDTA),
plasma (citrate), cell culture supernatant, and CSF sample types.
Saliva, urine, milk, cell extract, and tissue extract samples have not been tested with this kit.
50% serum from ten individual healthy human female and ten individual healthy human maledonors was measured in duplicate. All values were below the detectable range of the assay.

CROSS REACTIVITY--

Calculate the average absorbance value for the blank control (zero) standards. Subtract
the average blank control standard absorbance value from all other absorbance values.
Create a standard curve by plotting the average blank control subtracted absorbancevalue for each standard concentration (y-axis) against the target protein concentration(x-axis) of the standard. Use graphing software to draw the best smooth curve throughthese points to construct the standard curve.

 Note: Most microplate reader software or graphing software will plot these values andfit a curve to the data. A four parameter curve fit (4PL) is often the best choice; however,other algorithms (e.g. linear, semi-log, log/log, 4 parameter logistic) can also be tested todetermine if it provides a better curve fit to the standard values.
Determine the concentration of the target protein in the sample by interpolating the blankcontrol subtracted absorbance values against the standard curve. Multiply the resulting
value by the appropriate sample dilution factor, if used, to obtain the concentration oftarget protein in the sample.
Samples generating absorbance values greater than that of the highest standard should
be further diluted and reanalyzed. Similarly, samples which measure at absorbance values
less than that of the lowest standard should be retested in a less dilute form.

Recombinant proteins were prepared at 1,000 pg/mL and assayed for cross reactivity. No crossreactivity was observed for the following targets: Human IL-2, Human IL-4, Human IL-1 alpha,
Human IFN gamma, Human TNF beta, and Human TNF R1.

TYPICAL DATAINTERFERENCE

Typical standard curve – data provided for demonstration purposes only. A new standard
curve must be generated for each assay performed.

Recombinant Human TNF R1 was prepared at 1,000 pg/mL and was tested for interferencewith 1,000 pg/mL of recombinant Human TNF alpha. No interference was observed.

SPECIES REACTIVITY
1,000 pg/mL of recombinant primate, mouse, and rat TNF alpha were tested for reactivity.
35% primate reactivity was observed. No mouse or rat reactivity was observed.
Other species reactivity not determined.

Standard Curve Measurements
O.D 450 nm

Concentration(pg/mL)

1

2

Mean
O.D

0

0.061

0.068

0.065

15.63

0.127

0.123

0.125

CALIBRATION

31.25

0.189

0.206

0.197

The standard protein in this kit is the soluble form of TNF alpha calibrated to full-length highly

62.5

0.312

0.312

0.312

purified human TNF alpha. The NIBSC/WHO unclassified purified human TNF alpha preparation

125

0.555

0.521

0.538

250

1.065

1.030

1.048

500

1.925

1.941

1.933

1,000

3.362

3.161

3.262

12/154 was evaluated in this kit.
The dose response curve of the unclassified standard TNF alpha parallels the SimpleStep
standard curve. To convert sample values obtained with the SimpleStep Human TNF alpha kit
to approximate NIBSC 12/154 International units, use the equation below.
To convert sample values obtained with the kit to approximate NIBSC 12/154 units, use thefollowing equation: NIBSC (12/154) approximate value (IU/mL) = 0.094 x SimpleStep Human TNF
alpha value (pg/mL).

Table 1. Example of human TNF alpha standard curve in Sample Diluent NS. The TNF alphastandard curve was prepared as described in the Standard Preparation section. The tableshows raw data values.

TYPICAL SAMPLE VALUES
Sensitivity:
The calculated minimal detectable dose (MDD) is 4.32 pg/mL. The MDD was determined by
calculating the mean of zero standard replicates (n=24) and adding 2 standard deviations
then extrapolating the corresponding concentration.

RecoveryThree concentrations of TNF alpha were spiked in duplicate to the indicated biological matrix
to evaluate signal recovery in the working range of the assay.
Sample Type
Average % Recovery
2.5% Serum88
2.5% Plasma – Citrate
91
2.5% Plasma – EDTA
100
2.5% Plasma – Heparin
98
5% PHA-M treated PBMC Cell Culture
110Supernatant*
50% Cerebrospinal Fluid
83*Media is RPMI 1640 containing 10% fetal calf serum.

Range (%)
83 - 93
86 - 97
95 - 104
89 - 110
105 – 113

Recombinant TNF alpha was spiked into the following biological samples in a 2-fold dilution
series. Sample dilutions are made in Sample Diluent NS.
2.5%
2.5%
2.5%
2.5%
50%
Dilution
Human
Human
Human
Interpolated value
Human
Human
PlasmaPlasmaPlasmaFactorSerum
CSF(Citrate)(EDTA)(Heparin)
Undiluted

2

4

8

pg/mL

100

100

100

100

234.34244.59237.17

192.55% Expected value

100

104

101

95

98

pg/mL

119.50127.27123.94120.73110.67% Expected value

107

113

102

97

113

pg/mL

60.23

59.54

62.05

60.11

56.51

% Expected value

108

106

103

96

115

pg/mL

28.78

NL

NL

NL

NL

% Expected value

103

NL

NL

NL

NL

Precision

Undiluted

2

4

8% Expected value

100

pg/mL

355.55% Expected value

98

pg/mL

183.66% Expected value

101

pg/mL

94.78

% Expected value

104

392.65100

Linearity of dilution is determined based on interpolated values from the standard curve.
Linearity of dilution defines a sample concentration interval in which interpolated target
concentrations are directly proportional to sample dilution.

726.56499.11223.35

NL – Non-Linear

pg/mL

483.71

pg/mL

Linearity of Dilution

Native TNF alpha was measured in the following biological samples in a 2-fold dilution series.
Sample dilutions are made in Sample Diluent NS.
10% PHA-M
Dilution
Treated PBMCInterpolated value
FactorCell Culture
Supernatant

451.09% Expected value

80 - 8816447.20Mean coefficient of variations of interpolated values of TNF alpha from three concentrations of
PHA-M treated PBMC cell culture supernatant within the working range of the assay.
Intra-assay
Inter-assayN=83
CV (%)
2.5
3.1

Download our ELISA guide for technical hints, results, calculation, and troubleshooting tips:
www.abcam.com/protocols/the-complete-elisa-guide
For technical support contact information, visit: www.abcam.com/contactus
Copyright © 2023 Abcam, All Rights Reserved. All information / detail is correct at time of going to print.
Version 8 | 2023-10-10

DIRECTIONS FOR 384-WELL PLATE FORMAT:

Standard Preparation

Materials Supplied for 384-well format
Item

Quantity

Storage Condition

Always prepare a fresh set of standards for every use. Discard working standard dilutions after
use as they do not store well. The following section describes the preparation of a standard
curve
for
duplicatemeasurements(recommended).

Human TNF alpha Capture Antibody 10X
Human TNF alpha Detector Antibody 10X
Human TNF alpha Lyophilized Recombinant Protein
Antibody Diluent 4BR
Sample Diluent NS
Wash Buffer PT 10X
TMB Development Solution
Stop Solution
SimpleStep Pre-Coated 384-Well Microplate
Plate Seal

600 μL
600 μL
2 Vials
6 mL
100 mL
20 mL
2 x12 mL
2 x12 mL
384 wells
1

+4°C
+4°C
+4°C
+4°C
+4°C
+4°C
+4°C
+4°C
+4°C
+4°C

Materials Required, Not Supplied

1.

2.
3.
4.

Reconstitute the TNF alpha standard sample by adding the volume of Sample Diluent NS
indicated on the protein vial label. Hold at room temperature for 10 minutes. Mix
thoroughly and gently. This is the 10,000 pg/mL Stock Standard Solution.
Label eight tubes, Standards 1– 8.
Add 180 μL of Sample Diluent NS into tube number 1 and 75 μL of Sample Diluent NS into
numbers 2-8.
Use the Stock Standard to prepare the following dilution series. Standard #8 contains noprotein and is the Blank control:Standard #Dilution
SampleVolume to
Dilute (μL)

Volume ofDiluent (μL)

StartingConc.
(pg/mL)

Final
Conc.
(pg/mL)

1

Stock Standard

20

180

10,0001,000

2Standard#1

75

75

1,000

500

3

Standard#2

75

75

500

250

4

Standard#3

75

75

250

125

5

Standard#4

75

75

125

62.5

6

Standard#5

75

75

62.5

31.25

7

Standard#6

75

75

31.25

15.63

8

Blank Control

0

75

0

0

These materials are not included in the kit, but will be required to successfully utilize this assay:
Microplate reader capable of measuring absorbance at 450 or 600 nm in a 384-well plate.
Deionized water.
Multi- and single-channel pipettes.
Tubes for standard dilution.
Plate shaker for all incubation steps.
Optional: Phenylmethylsulfonyl Fluoride (PMSF) (or other protease inhibitors).
Optional: Automated liquid handler.

Reagent Preparation
Equilibrate all reagents to room temperature (18-25°C) prior to use. The kit contains enoughreagents for one full plate. The sample volumes below are sufficient for running all 384 wells;
adjust volumes as needed for the number of samples and dilution scheme for your experiment.
Prepare only as much reagent as is needed on the day of the experiment. Capture anddetector antibodies have only been tested for stability in the provided 10X formulations.
1X Wash Buffer PT: Prepare 1X Wash Buffer PT by diluting Wash Buffer PT 10X with deionizedwater. To make 50 mL 1X Wash Buffer PT combine 5 mL Wash Buffer PT 10X with 45 mL deionizedwater. Mix thoroughly and gently.
Antibody Cocktail: Prepare Antibody Cocktail by diluting the capture and detector antibodies
in Antibody Diluent 4BR . To make 6 mL of the Antibody Cocktail combine 600 µL 10X Capture
Antibody and 600 µL 10X Detector Antibody with 4.8 mL Antibody Diluent 4BR . Mix thoroughly
and gently.

Plate Preparation
The 384-well plate included with this kit are supplied ready to use. It is not necessary to rinse theplate prior to adding reagents.
For each assay performed, a minimum of two wells must be used as the zero control.
For statistical reasons, we recommend each sample should be assayed with a minimum of two
replicates (duplicates).
Differences in well absorbance or “edge effects” have not been observed with this assay.

Assay Procedure for 384-well plate format
Equilibrate all materials and prepared reagents to room temperature prior to use.
We recommend that you assay all standards, controls and samples in duplicate.
1.
2.
3.
4.
5.

6.

Prepare all reagents, working standards, and samples as directed in the previous sections.
Add 12.5 µL of all sample or standard to appropriate wells.
Add 12.5 µL of the Antibody Cocktail to each well.
Seal the plate and incubate for 1 hour at room temperature on a plate shaker set to
700 rpm.
Wash each well with 3 x 100 µL 1X Wash Buffer PT. Wash by aspirating or decanting fromwells then dispensing 100 µL 1X Wash Buffer PT into each well. Wash Buffer PT should remain
in wells for at least 10 seconds. Complete removal of liquid at each step is essential forgood performance. After the last wash invert the plate and tap gently against clean
paper towels to remove excess liquid.
Add 25 µL of TMB Development Solution to each well and incubate for 10 minutes in thedark on a plate shaker set to 700 rpm.

Given variability in laboratory environmental conditions, optimal incubation time may vary
between 5 and 20 minutes.

7.

8.

Note: The addition of Stop Solution will change the color from blue to yellow and enhance
the signal intensity about 3X. To avoid signal saturation, proceed to the next step before
the high concentration of the standard reaches a blue color of O.D.600 equal to 1.0.
Add 25 µL of Stop Solution to each well. Shake plate on a plate shaker for 1 minute to mix.
Record the OD at 450 nm. This is an endpoint reading. Proper mixing of the Stop Solution isrequired for proper measurement.
Alternative to 6 – 7: Instead of the endpoint reading at 450 nm, record the developmentof TMB Substrate kinetically. Immediately after addition of TMB Development Solution
begin recording the blue color development with elapsed time in the microplate reader
prepared with the following settings:
Mode

Kinetic

Wavelength:

600 nmTime:

up to 20 min

Interval:

20 sec - 1 min

Shaking:

Shake between readings

Note that an endpoint reading can also be recorded at the completion of the kinetic
read by adding 25 µL Stop Solution to each well and recording the OD at 450 nm.
Download our ELISA guide for technical hints, results, calculation, and troubleshooting tips:
www.abcam.com/protocols/the-complete-elisa-guide
For technical support contact information, visit: www.abcam.com/contactus

Copyright © 2023 Abcam, All Rights Reserved. All information / detail is correct at time of going to print.
Version 8 | 2023-10-10