ab136952 – sTL1A
Human ELISA
Development Set
(Reagents for 5 x 96 well
plates)
Instructions for Use
For quantitative detection of sTL1A in Human biological fluids.
This product is for research use only and is not intended for diagnostic
use.
Version 1 Last Updated 15 January 2014
Table of Contents
INTRODUCTION
1.
BACKGROUND
2.
ASSAY SUMMARY
2
3
GENERAL INFORMATION
3.
PRECAUTIONS
4.
STORAGE AND STABILITY
5.
MATERIALS SUPPLIED
6.
MATERIALS REQUIRED, NOT SUPPLIED
7.
LIMITATIONS
8.
TECHNICAL HINTS
4
5
5
6
6
7
ASSAY PREPARATION
9.
REAGENT PREPARATION
10. STANDARD PREPARATIONS
11. SAMPLE COLLECTION AND STORAGE
12. PLATE PREPARATION
8
9
11
12
ASSAY PROCEDURE
13. ASSAY PROCEDURE
13
DATA ANALYSIS
14. CALCULATIONS
15. TYPICAL DATA
16. TYPICAL SAMPLE VALUES
17. ASSAY SPECIFICITY
15
16
17
18
RESOURCES
18.
19.
TROUBLESHOOTING
NOTES
Discover more at www.abcam.com
19
20
1
INTRODUCTION
1. BACKGROUND
Abcam’s sTL1A in vitro Human ELISA (Enzyme-Linked
Immunosorbent Assay) kit is designed for the accurate quantitative
measurement of sTL1A in Human biological fluids.
A monoclonal antibody specific for sTL1A is coated onto 96-well
plates. Samples and standards of sTL1A are then pipetted into the
wells for binding to the coated antibody. After extensive washing to
remove unbound compounds, the bound sTL1A is recognized by the
addition of a biotinylated monoclonal antibody specific for sTL1A. After
removal of excess biotinylated antibody, streptavidin-peroxidase is
added. Following a final washing, peroxidase activity is quantified
using the substrate 3,3’,5,5’- tetramethylbenzidine (TMB). The intensity
of the color reaction is measured at 450 nm after the addition of 1N
HCl and is directly proportional to the concentration of sTL1A in the
samples.
TL1A, a ligand belonging to the tumor necrosis factor (TNF) family
(also called TNFSF15), is expressed predominantly by endothelial
cells, but is also found on lymphocytes, plasma cells and monocytes.
TL1A is up regulated by the proinflammatory cytokines TNF and IL-1.
On activated T cells, TL1A functions specifically via its surface-bound
receptor DR3, (a member of the death-domain containing TNF
receptor family) to promote cell survival and secretion of
proinflammatory cytokines. The decoy receptor 3 (DcR3), a soluble
protein of the tumor necrosis factor receptor (TNFR) superfamily
blocks the action of TL1A. TL1A, like TNF, can be released to circulate
as a homotrimeric soluble form. Activation of DR3 by TL1A induces the
formation of a signaling complex containing TRADD, TRAF2, and RIP
and activates the NF-kappaB and the ERK, JNK, and p38
mitogenactivated protein kinase pathways. The TL1A/DR3 pathway
may be involved in atherosclerosis via the induction of proinflammatory cytokines/chemokines and seems to play an important
role in Th1-mediated intestinal diseases, such as Crohn’s disease.
Discover more at www.abcam.com
2
INTRODUCTION
2. ASSAY SUMMARY
Coat Capture Antibody onto 96 well
plates.
Add standards and samples to
appropriate wells. Incubate for 1 hour.
Wash each well and add prepared
Detection Antibody to appropriate
wells.
Wash each well and add HRPStreptavidin conjugate to each well to
appropriate wells. Incubate at room
temperature.
After washing, add TMB substrate to
each well. Incubate at room
temperature. Add 1N HCL to each
well. Read immediately.
Discover more at www.abcam.com
3
GENERAL INFORMATION
3. PRECAUTIONS
Please read these instructions carefully prior to beginning the
assay.

Some kit components contain azide, which may react with lead or
copper plumbing. When disposing of reagents always flush with
large volumes of water to prevent azide build-up

Stop Solution is a solution of trisodium phosphate. This solution is
caustic; care should be taken in use

We test this kit’s performance with a variety of samples, however it
is possible that high levels of interfering substances may cause
variation in assay results
Discover more at www.abcam.com
4
GENERAL INFORMATION
4. STORAGE AND STABILITY
Store kit at +4°C immediately upon receipt, apart from the sTL1A
Horseradish Peroxidase Conjugate and sTL1A Standard, which
should be stored at -20°C. Avoid multiple freeze-thaw cycles.
Refer to list of materials supplied for storage conditions of individual
components.
5. MATERIALS SUPPLIED
120 µL
Storage
Condition
(Before
Preparation)
-20ºC
Standard Lyophilized (STD)
2 µg
-20ºC
Detection Antibody (DET) (1 mg/mL)
60 µL
-20ºC
Item
Coating Antibody (COAT) (1 mg/mL)
Discover more at www.abcam.com
Amount
5
GENERAL INFORMATION
6. MATERIALS REQUIRED, NOT SUPPLIED
These materials are not included in the kit, but will be required to
successfully utilize this assay:

Standard microplate reader - capable of reading at 450 nm,
preferably with correction between 570 and 590 nm.

Automated plate washer (optional)

Adjustable pipettes and pipette tips. Multichannel pipettes are
recommended when large sample sets are being analyzed

Eppendorf tubes

Absorbent paper for blotting

PBS: 100 mM PO4, pH 7.1, 75 mM NaCl

1N Hydrochloric acid (HCl)

Tetramethylbenzidine substrate (TMB)

HRP-Labeled Streptavidin (SA-HRP)

HPLC grade water
7. LIMITATIONS

Assay kit intended for research use only. Not for use in diagnostic
procedures

Do not mix or substitute reagents or materials from other kit lots or
vendors. Kits are QC tested as a set of components and
performance cannot be guaranteed if utilized separately or
substituted
Discover more at www.abcam.com
6
GENERAL INFORMATION
8. TECHNICAL HINTS

Standards can be made up in either glass or plastic tubes

Pre-rinse the pipette tip with the reagent, use fresh pipette tips for
each sample, standard and reagent

Pipette standards and samples to the bottom of the wells

Add the reagents to the side of the well to avoid contamination

Be sure no sodium azide is present in this assay, as this inhibits
HRP enzyme activity

Once reagents have been added to the plate, DO NOT let the
plate dry at any time during the assay.

It is recommended that all standards and samples be assayed in
duplicate.

Avoid microbial contamination of reagents and equipment.
Automated plate washers can become contaminated thereby
causing assay variability. Buffers containing a large quantity of
protein should be made under sterile conditions and stored at 28°C or be prepared fresh daily.

This kit uses break-apart microtiter strips, which allow the user to
measure as many samples as desired. Unused wells must be kept
desiccated at 4°C in the sealed bag provided. The wells should be
used in the frame provided

Prior to addition of substrate, ensure that there is no residual wash
buffer in the wells. Any remaining wash buffer may cause variation
in assay results

This kit is sold based on number of tests. A ‘test’ simply
refers to a single assay well. The number of wells that contain
sample, control or standard will vary by product. Review the
protocol completely to confirm this kit meets your
requirements. Please contact our Technical Support staff with
any questions
Discover more at www.abcam.com
7
ASSAY PREPARATION
9. REAGENT PREPARATION
Equilibrate all reagents and samples to room temperature (18 - 25°C)
prior to use.
9.1
1X Coating Antibody
Dilute the stock 1 mg/mL Coating Antibody (COAT) to 1X in
PBS. To generate 10 mL of the 1X Coating Antibody, dilute
20 μL of the 1 mg/mL Coating Antibody in 9,980 μL of PBS.
9.2
1X Detection Antibody
To generate the 1X Detection Antibody dilute the stock
1 mg/mL Detection Antibody (DET) in PBS. To generate
10 mL of 1X Detection Antibody, dilute 10 μL of the 1 mg/mL
Detection Antibody in 9,990 μL of PBS.
9.3
Blocking Buffer
Prepare the Blocking Buffer using the following recipe:
1% BSA in PBS
9.4
ELISA Buffer
Prepare the ELISA Buffer using the following recipe:
1% BSA and 0.1% 20 in PBS Tween-20, pH 7.4
9.5
Wash Buffer
Prepare the Wash Buffer using the following recipe: 0.1%
Tween-20 in PBS
9.6
Streptavidin-HRP
Prepare
the
Streptavidin-HRP
according
to
the
manufactures instructions. Dilute in ELISA buffer if required.
Discover more at www.abcam.com
8
ASSAY PREPARATION
10. STANDARD PREPARATIONS
Prepare serially diluted standards immediately prior to use. Always
prepare a fresh set of standards for every use. Reconstitution of the
sTL1A standard should be prepared no more than 1 hour prior to the
experiment. Diluted standards should be used within 60 minutes of
preparation.
10.1 Reconstitute the sTLA1 Standard by adding 100 µL ELISA
Buffer by pipette. Mix thoroughly and gently. This is the
200 ng/mL Stock Standard (see table below). Aliquot and
store any unused standard at -20ºC. Avoid freeze/thaw
cycles.
10.2 Label eight tubes with numbers 1 – 8.
10.3 Add 150 μL ELISA Buffer into tube numbers 2 - 8.
10.4 Prepare a 2.5 ng/mL Standard 1 by adding 3.75 μL of the
200 ng/mL Stock Standard Solution and 296.25 µL of ELISA
Buffer to tube 1. Mix thoroughly and gently.
10.5 Prepare Standard 2 by transferring 150 μL
Standard #1 to tube 2. Mix thoroughly and gently.
from
10.6 Prepare Standard 3 by transferring 150 μL from Standard 2
to tube 3. Mix thoroughly and gently.
10.7 Using the table below as a guide, repeat for tubes 4 through
7.
10.1 Standard 8 contains no protein and is the Blank control.
Discover more at www.abcam.com
9
ASSAY PREPARATION
Standard
#
Sample to
Dilute
Volume to
Dilute
(µL)
1
2
3
4
5
6
7
8 (Blank)
Stock
Standard #1
Standard #2
Standard #3
Standard #4
Standard #5
Standard #6
none
3.75
150
150
150
150
150
150
-
Discover more at www.abcam.com
Volume
of
Diluent
(µL)
296.25
150
150
150
150
150
150
150
Starting
Conc.
(ng/mL)
Final
Conc.
(ng/mL)
200
2.5
1.25
0.625
0.312
0.156
0.078
-
2.5
1.25
0.625
0.312
0.156
0.078
0.039
0
10
ASSAY PREPARATION
11. SAMPLE COLLECTION AND STORAGE

If measuring serum or cell culture supernatant, dilute samples in
ELISA buffer.

To reduce interference from rheumatoid factor (RF) in the serum,
Ig can be cleared by treating serum dilutions with 0.05 volume of
protein G-Sepharose twice for one hour at 4°C before pelleting the
beads and collecting supernatants (precleared sera).

Store samples to be assayed within 24 hours at 2 - 8°C. For longterm storage, aliquot and freeze samples at -20°C. Avoid repeated
freeze-thaw cycles.
Discover more at www.abcam.com
11
ASSAY PREPARATION
12. PLATE PREPARATION

For each assay performed, a minimum of 2 wells must be used as
blanks, omitting primary antibody from well additions

For statistical reasons, we recommend each sample should be
assayed with a minimum of two replicates (duplicates)
12.1. Add 100 µL of the 1X Capture Antibody into the appropriate
number of wells in the 96 well microtiter plates. Seal the
plate and incubate overnight at 4ºC.
12.2. Aspirate the coated wells and add 400 μL of Wash Buffer
using a multichannel pipette or autowasher. Repeat the
process for a total of four washes. Complete removal of
liquid at each step is essential for good performance. After
the last wash, remove any remaining Wash Buffer by
aspirating or by inverting the plate and blotting it against
clean paper towels.
12.3. Add 200 µL Blocking Buffer to each well. Seal the plate and
incubate for at least for 2 – 4 hours at room temperature.
12.4. Repeat Step 12.2, but do not remove the last wash until
ready to begin Assay Procedure, Step 13.2.
Discover more at www.abcam.com
12
ASSAY PROCEDURE
13. ASSAY PROCEDURE

Equilibrate all materials and prepared reagents to room
temperature prior to use

It is recommended to assay all standards, controls and
samples in duplicate
13.1
Prepare all reagents, working standards, and samples as
directed in the previous sections.
13.2
Add a total of 100 μL/well diluted serum, diluted culture
supernatant or Standard Protein serial dilutions in ELISA
Buffer to the plate.
13.3
Cover the plate with plastic film and incubate for one hour
at room temperature.
13.4
Aspirate the coated wells and add 400 μL of Wash Buffer
using a multichannel pipette or autowasher. Repeat the
process for a total of four washes. After the last wash,
complete removal of liquid is essential for good
performance.
13.5
Add 100 μL/well of the 1X Detection Antibody.
13.6
Cover the plate with plastic film and incubate for one hour
at room temperature.
13.7
Wash as described in step 13. 4 for a total of four washes.
13.8
Add 100 μL to each well of the Streptavidin-HRP.
13.9
Cover the plate with plastic film and incubate for 30 min at
room temperature.
13.10 Aspirate the coated wells and add 400 μL of Wash Buffer
using a multichannel pipette or autowasher. Repeat the
process for a total of four washes. After the last wash,
complete removal of liquid is essential for good
performance.
13.11 Add TMB Substrate to each well. An incubation of 15 – 30
minutes at room temperature is recommended.
Discover more at www.abcam.com
13
ASSAY PROCEDURE
13.12 Stop the reaction by adding 100 μL of 1N HCl stop solution
into each well. Tap the plate gently to ensure thorough
mixing.
13.13 Blank the plate reader against the blank wells, read the
O.D. absorbance at 450 nm, preferably with correction
between 570 and 590 nm. If the plate reader is not able to
be blanked against the blank wells, manually subtract the
mean optical density of the blank wells from all readings.
Discover more at www.abcam.com
14
DATA ANALYSIS
14. CALCULATIONS

Average the duplicate readings for each standard control and
sample and subtract the average blank value.

Generate the standard curve by plotting the average absorbance
obtained for each standard concentration on the vertical (Y) axis
vs. the corresponding hTL1A concentration (ng/ml) on the
horizontal axis (see TYPICAL DATA).

Calculate results using graph paper or curve-fitting statistical
software. We recommend that the data be handled by an
immunoassay software package utilizing a four parameter logistic
curve fitting program. The amount of hTL1A in each sample is
determined by interpolating for the absorbance value (Y-axis)
using the standard curve.

If the test sample was diluted, multiply the interpolated value by
the dilution factor to calculate ng/ml of Human TL1A in the sample
.
Discover more at www.abcam.com
15
DATA ANALYSIS
15. TYPICAL DATA
TYPICAL STANDARD CURVE – Data provided for demonstration
purposes only. A new standard curve must be generated for each
assay performed. The following data are obtained using the different
concentrations of standard as described in this protocol:
Conc.
(ng/mL)
Mean OD
0
0.093
0.039
0.124
0.078
0.127
0.156
0.167
0.312
0.250
0.625
0.400
1.25
0.714
2.5
1.495
Discover more at www.abcam.com
16
DATA ANALYSIS
16. TYPICAL SAMPLE VALUES
SENSITIVITY –
The sensitivity of sTL1A using this Abcam Human ELISA kit was found
to be 20 pg/mL (range 0 to 2.5 ng/mL).
Discover more at www.abcam.com
17
DATA ANALYSIS
17. ASSAY SPECIFICITY
CROSS REACTIVITY –
The monoclonal antibodies used in this detection Set are specific for
measurement of endogenous and recombinant Human sTL1A. They
do not cross-react with mouse sTL1A.
Discover more at www.abcam.com
18
RESOURCES
18. TROUBLESHOOTING
Problem
Poor
standard
curve
Low Signal
Samples
give higher
value than
the highest
standard
Cause
Solution
Inaccurate pipetting
Check pipettes
Improper standards
dilution
Prior to opening, briefly spin the
stock standard tube and dissolve
the powder thoroughly by gentle
mixing
Incubation times too
brief
Ensure sufficient incubation times;
change to overnight
standard/sample incubation
Inadequate reagent
volumes or improper
dilution
Check pipettes and ensure correct
preparation
Starting sample
concentration is too
high.
Dilute the specimens and repeat
the assay
Plate is insufficiently
washed
Review manual for proper wash
technique. If using a plate washer,
check all ports for obstructions
Contaminated wash
buffer
Prepare fresh wash buffer
Improper storage of
the kit
Store the all components as
directed.
Large CV
Low
sensitivity
Discover more at www.abcam.com
19
RESOURCES
19. NOTES
Discover more at www.abcam.com
20
RESOURCES
Discover more at www.abcam.com
21
RESOURCES
Discover more at www.abcam.com
22
UK, EU and ROW
Email: technical@abcam.com | Tel: +44-(0)1223-696000
Austria
Email: wissenschaftlicherdienst@abcam.com | Tel: 019-288-259
France
Email: supportscientifique@abcam.com | Tel: 01-46-94-62-96
Germany
Email: wissenschaftlicherdienst@abcam.com | Tel: 030-896-779-154
Spain
Email: soportecientifico@abcam.com | Tel: 911-146-554
Switzerland
Email: technical@abcam.com
Tel (Deutsch): 0435-016-424 | Tel (Français): 0615-000-530
US and Latin America
Email: us.technical@abcam.com | Tel: 888-77-ABCAM (22226)
Canada
Email: ca.technical@abcam.com | Tel: 877-749-8807
China and Asia Pacific
Email: hk.technical@abcam.com | Tel: 108008523689 (中國聯通)
Japan
Email: technical@abcam.co.jp | Tel: +81-(0)3-6231-0940
www.abcam.com | www.abcam.cn | www.abcam.co.jp
Copyright © 2013 Abcam, All Rights Reserved. The Abcam logo is a registered trademark.
All information / detail is correct at time of going to print.
RESOURCES
23