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ELISA kit
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INFO @ucytech.com
www.ucytech.com

5-plate format

For research use only.

Not for use in diagnostic or therapeutic procedures.
Contents
Introduction  4
Principle of the test  5
Warnings and precautions  6
Contents of the kit  7
Storage and stability  8
Materials and equipment (required but not provided)  9
Specimen collection and handling  9
Preparation solutions and reagents  10
Sample preparation  11
Preparing the standard curve  12
Directions for washing  13
Data analysis  13
Assay procedure  14
Troubleshooting  15
Technical assistance

Abbreviations
BSA Bovine serum albumin
CSB Cytokine stabilization buffer
ELISA Enzyme-linked immunosorbent assay
G-CSF Granulocyte Colony Stimulating Factor
GM-CSF Granulocyte-Macrophage Colony Stimulating Factor
HRP Horse Radish Peroxidase
IFN Interferon
IL Interleukin
L Liter
MCP Monocyte Chemoattractant Protein
min minute(s)
NCCLS National Committee for Clinical Laboratory Standards
OD Optical density
PB Phosphate buffer
PBS Phosphate buffered saline
RT Room temperature (temperature between 20 ºC and 26 ºC)
SPP Streptavidin-HRP
Std Standard dilution
TMB 3,3’,5,5’-Tetramethylbenzidine
TNF Tumor necrosis factor

2
This manual applies to the following U-CyTech ELISA kits

Human Old World New World Mouse Rat

Monkey Monkey
IFN-γ CT201A CT141A CT340A CT301A CT071A
IL-1β CT526A CT139A
IL-2 CT202A CT142A CT344A CT309A
IL-4 CT203A CT143A CT306A CT073A
IL-5 CT204A CT144A CT296A
IL-6 CT205A CT145A CT346A CT299A
IL-7 CT523A
IL-8 CT212A CT151A
IL-10 CT206A CT146A CT307A
IL-12/23p40 CT149A CT345A
IL-12p70 CT210A
IL-13 CT208A CT147A CT341A
IL-17A CT516A CT501A CT343A
IL-17F CT518A CT503A
IL-17AF CT515A
IL-21 CT530A
IL-23 CT517A CT502A
IL-27 CT524A
IL-29 CT525A
IL-31 CT520A
IL-33 CT519A
IP-10 CT522A CT157A
Angiopoietin-2 CT527A CT158A
G-CSF CT389A CT155A
GM-CSF CT200A CT140A
Granzyme B CT211A
MCP-1 CT521A CT156A
MICB CT528A
Perforin CT391A CT154A
VEGF-A CT529A
TNF-α CT209A CT148A CT342A CT303A CT075A

Overview of catalogue numbers of U-CyTech ELISA kits.

3
Introduction
Cytokines, chemokines and granzymes are a group of signaling proteins that affect the behavior
of cells when released. They are critically involved in various physiological processes such
as immune regulation, cell differentiation, cell proliferation, chemotaxis and cell apoptosis.
These signaling proteins are produced by a variety of different cell types in many vertebrate
species and are active at very low concentrations mostly in the picogram to femtogram range.
The enzyme-linked immunosorbent assay (ELISA) is one of the primary and most popular
methods to detect and measure these signaling proteins. The ELISA test is rapid, simple to
perform and is one of the most sensitive and reliable technologies available.
U-CyTech has developed various high-quality ELISA kits for the detection of cytokines,
chemokines and granzymes for human, monkey (including macaques and marmoset), mouse
and rat. These assays are widely applied in different research fields, including cancer research,
vaccine development, infectious diseases, autoimmune diseases, organ transplantation
and parasitology. Hundreds of peer-reviewed publications describe the successful use of
U-CyTech’s ELISA systems in different biological fluids.

Please, find references of studies in different research areas using our ELISA kits on:
www.ucytech.com/ELISA of www.ucytech.com/references.

4
Principle of the test
U-CyTech’s ELISA kits are simple and sensitive sandwich immunoassays for the determination
of cytokine, chemokine and granzyme levels in biological fluids such as cell culture
supernatant, plasma or serum.
The assays utilize a coating antibody specific for the analyte of interest (e.g. cytokine,
chemokine or granzyme) coated on the wells of a 96-well microtiter plate. The wells are
washed and blocked. Standards and samples are added to the wells and any analyte present
binds to the immobilized coating antibody.
After washing off the excess and unbound materials, the bound analyte is allowed to associate
with a biotinylated detection antibody. The wells are washed again and a streptavidin-HRP
(SPP) conjugate is added to the antibody-antigen-antibody complex. After another wash,
a chromogenic substrate (TMB) is introduced, which produces a blue-colored product of
which the intensity is related to the amount of analyte in the sample. A sulfuric acid solution
is added to stop the enzymatic reaction (changing the color to yellow) and OD is read at
450 nm.

Add coating Block Add samples and Add biotinylated

antibody standards detection antibody

wash wash

wash

wash

Add stop solution Add substrate Add conjugate

5
Warnings and precautions
• This kit is designed for research use only, and not for use in diagnostic or therapeutic
procedures.
• When cytokine, chemokine or granzyme levels are determined in blood components or
other biological materials, then please note that all these materials should be considered
as potentially infectious and handled with the usual precautions under Bio-Hazard
conditions. Follow universal precautions such as established by the US government
agencies, Centers for Disease Control and Prevention and Occupational Safety and Health
Administration, when handling and disposing of (potentially) infectious waste.

Hazard information
All kit components are not classified as dangerous according to Regulation (EC) no. 1272/2008 and
Directive 67/548/EEC or 1999/45/EC and their amendments.
Please find the Material Safety Data Sheet on www.ucytech.com/manuals.

6
Contents of the kit

Items Quantity (5-plate format) Storage conditions

Coating antibody* 1 vial 4 ºC

Standard* 5 vials 4 ºC
Biotinylated detection antibody* 1 vial 4 ºC
SPP conjugate* 1 vial ≤-20 ºC in the dark
BSA stock solution (10%) 2 vials (2 x 12 ml) 4 ºC
Cytokine stabilization buffer (CSB)** 1 vial (5 ml) 4 ºC
Tween-20 1 vial (5 ml) RT in the dark
TMB substrate solution 2 vials (2x 30 ml) 4 ºC in the dark
Stop solution (0.175 M H2SO4) 2 vials (2x 30 ml) 4 ºC
ELISA plates 8 plates RT
Adhesive cover slips 10 slips RT

* Lyophilized.
** For use in serum and plasma samples only, see section “Sample preparation” on page 11.

7
Storage and stability
Coating antibody and biotinylated detection antibody
The vials with lyophilized coating and biotinylated detection antibody can be safely stored
at 4 ºC until the expiry date (indicated on the vials). After reconstitution, the antibodies are
stable for at least 12 months at 4 ºC when kept sterile. However, it is recommended to divide
the reconstituted antibody solutions into small aliquots for single use. These aliquots should
be stored at ≤-20 ºC (stable for at least two years).

Standard
The vials with lyophilized standard can be safely stored at 4 ºC until the expiry date (indicated
on the vials). These vials are for single use only.

SPP conjugate
The vial with lyophilized SPP conjugate is stable until the expiry date (indicated on the
vial) when stored at ≤-20 ºC in the dark. After reconstitution, the reagent is stable for at
least 2 months at 4 ºC when kept sterile and protected from light. However, it is strongly
recommended to divide the solution into small aliquots for single use. These aliquots should
be stored at ≤-20 ºC in the dark (stable for at least one year).

TMB substrate solution

The ready-to-use TMB substrate solution should be stored at 4 ºC and is stable until the expiry
date (indicated on the vial). Avoid exposure to light, heat and contamination with metal ions
or peroxidase.

BSA stock solution and Cytokine stabilization buffer

The vials with BSA stock solution and Cytokine stabilization buffer can be safely stored at 4 ºC
until the expiry date (indicated on the vial). After opening, these solutions are stable for at
least 6 months when kept sterile.

Tween-20
Tween-20 can safely be stored at RT and is stable until the expiry date (indicated on the vial).

Stop solution
The ready-to-use Stop solution can safely be stored at 4 ºC and is stable until the expiry date
(indicated on the vial).

8
Materials and equipment (required but not provided)
• PBS (pH 7.4; ingredients: Na2HPO4·2H2O, KH2PO4, NaCl and distilled water).
Alternatively, use commercially available liquid PBS (pH 7.4) from Life Technologies
(cat. no. 10010-015) or other suppliers.
• Sterile distilled water.
• Pipetting devices for the accurate delivery of volume required for the assay performance.
• Tubes and containers/plates to make the solutions.
• 37 ºC incubator.
• Plate washer: automated or manual (squirt bottle, manifold dispenser).
• Reading device for microtiter-plate (wavelength set to 370, 450 or 655 nm).

Specimen collection and handling

Specimens should be clear, non-hemolyzed and non-lipemic. Excessive hemolysis and the
presence of large clots or microbial growth in the sample may interfere with the performance
of the test.

• Cell culture supernatant: remove any particulate matter by centrifugation.

• Serum: use a clot tube and allow sample to clot for 30-45 min at RT, then centrifuge for
10-15 min at 1,000-2,000 x g (RT) and collect serum immediately.
• Plasma: collect plasma by using anticoagulant, such as EDTA or heparin. Mix well
immediately after collection. Centrifuge for 10-15 min at 1,000-2,000 x g (RT) and collect
plasma.

Samples should be aliquoted and stored frozen at ≤-20 ºC to prevent cytokine degradation.
If samples are run within 24 hours, they may be stored at 2-8 ºC. Avoid repeated freeze-
thaw cycles. Do not heat serum or plasma samples. Prior to assay, frozen samples should be
completely thawed and mixed well.
Note: Specimen collection from humans and non-human primates should be carried out in
accordance with NCCLS document M29-T2, “Protection of laboratory workers from infectious
diseases transmitted by blood and tissue”.

9
Preparation solutions and reagents
PBS
PB stock: dissolve 96.0 g of Na2HPO4·2H2O plus 17.5 g of KH2PO4 in 1 L distilled water, adjust
pH to 7.4 and filtrate solution (0.2 µm). Store solution at RT (stable for at least 6 months
when kept sterile).
PBS: add 10 ml of the PB stock and 8.8 g of NaCl to 1 L distilled water. It is strongly recom-
mended to prepare PBS freshly each day. Alternatively, when PBS is prepared in advance, the
solution should be filter sterilized (0.2 µm) or autoclaved.

Wash buffer
PBS containing 0.05% Tween-20 (add 0.5 ml of Tween-20 to 1 L PBS). The volume is depending
on the washing procedure (manual or automatic washing).

Blocking buffer
PBS containing 1% BSA. For one ELISA plate: mix 2 ml BSA stock solution (10%) gently but
thoroughly with 18 ml PBS.

Dilution buffer
PBS containing 0.5% BSA and 0.05% Tween-20. You can prepare this buffer at once for 5 ELISA
plates by making at least 250 ml under sterile conditions. Add 12.5 ml of BSA stock solution
(10%) and 125 µl of Tween-20 to 250 ml PBS, mix gently and store at 4 ºC. This solution will
be stable for at least one month when kept sterile.
For one ELISA plate, 20 ml of Dilution buffer is needed for detection and conjugate solutions,
and at least 20 ml for standards and samples (this volume will depend on the number of
sample dilutions).

Standard
Reconstitute the lyophilized standard by injecting 500 μl of sterile distilled water into the
vial. Mix the solution gently for approximately 15 seconds and allow it to stand for 5 min at
RT. Avoid vigorous shaking. Thereafter, the reconstituted Standard is placed on melting ice
and is immediately (preferentially within one hour) diluted as described in “Preparing the
standard curve” on page 12.
Note: the quantity (expressed in ng/vial) of the Standard is indicated on the vial and is
variable for each kit and batch. After reconstitution, the concentration can be calculated as
follows: divide the quantity (indicated on the vial) by the volume used for reconstitution.
For example, the concentration of a standard containing 5 ng/vial will be 10 ng/ml (= 10,000
pg/ml) after reconstitution in 0.5 ml distilled water.

10
Coating antibody
Reconstitute the lyophilized antibody by injecting 250 µl of sterile distilled water into the vial.
Mix the solution gently for approximately 15 seconds and allow it to stand for 5 min at RT.
Avoid vigorous shaking. For one ELISA plate: 50 µl is gently but thoroughly mixed with 5 ml PBS.
Note: Do not use commercially available PBS tablets for the preparation of the coating
solution (the filler in the tablets interferes with the coating process).

Biotinylated detection antibody

Reconstitute the lyophilized antibody by injecting 500 µl of sterile distilled water into the vial.
Mix the solution gently for approximately 15 seconds and allow it to stand for 5 min at RT. Avoid
vigorous shaking.
For one ELISA plate: 100 µl is gently and thoroughly mixed with 10 ml Dilution buffer.

SPP conjugate
Reconstitute the contents of the vial by injecting 500 µl of sterile distilled water into the vial.
Mix the solution gently for approximately 15 seconds and allow it to stand for 5 min at 4 ºC in
the dark. Avoid vigorous shaking.
For one ELISA plate: 100 µl is gently and thoroughly mixed with 10 ml Dilution buffer.

TMB substrate solution (ready-to-use)

Bring TMB substrate solution to RT prior to use.

Stop solution (ready-to-use)

Bring Stop solution to RT prior to use.

Sample preparation
Dilute samples in Dilution buffer (at least 1:1). It is recommended to analyze a series of
dilutions of the sample to ensure that sample measurements fall within the assay range (see
also ”Data analysis” on page 13).
When measuring cytokines in serum or plasma, add 1/20 volume of CSB (ready-to-use) to
the pure serum or plasma sample (CSB is not required for other samples such as cell culture
supernatant) before further dilution in Dilution buffer. CSB inhibits the degradation of cytokines.
It is recommended to test samples in triplicate.

11
Preparing the standard curve
With a standard curve the analyte concentration in the unknown samples can be determined.
The standard curve is generated from the data of 7 two-fold serial dilutions (Std 1-7) of
the Standard. The recommended assay range for each specific ELISA kit can be found in the
Typical data sheet on www.ucytech.com. It is recommended to test the standard dilutions
(Std 1-7) in triplicate.

• Take 8 tubes and add 400 µl Dilution buffer to 7 of these tubes (Std 2 till Std 7 and Blank).
• Prepare in the remaining tube (Std 1) the highest concentration to be used in the standard
curve (see Typical data sheet) by mixing an appropriate volume of Standard with Dilution
buffer. The final volume of Std 1 should be 800 µl. Allow the mixture to stand for at least
15 seconds before using in further dilutions.
• Perform two-fold serial dilutions: transfer 400 µl diluted standard from Std 1 to the next
tube (Std 2), mix well and repeat this step until Std 7.

First add 400 μl dilution buffer

400 μl 400 μl 400 μl 400 μl 400 μl 400 μl

Std 1 Std 2 Std 3 Std 4 Std 5 Std 6 Std 7 Blank

Standard

Notes:
• If less than 10 μl of the Standard is needed to make Std 1 it is recommended to dilute the
Standard 10 times in Dilution buffer (mix 10 μl Standard with 90 μl Dilution buffer) and
use this to make Std 1.
• A standard curve, including blank, should be run on each ELISA plate.
• Use vials with Standard only once.
• It is recommended to test standard dilutions in triplicate.
• Standard dilutions should be used as soon as possible (preferentially within one hour).
• Depending on the biological origin of the unknown samples, also other appropriate
dilution buffers may be used for the preparation of the standard curve, depleted with
the endogenous protein to be quantified (e.g. cell culture medium, serum).

12
Directions for washing
• Incomplete washing of the wells will adversely affect the assay. All washing steps should
be performed with Wash buffer.
• Washing can be performed manually as follows: completely aspirate the liquid from all
wells by gently lowering the tip of an aspiration device into each well (without touching
the bottom). After aspiration, fill the wells with at least 250 µl Wash buffer and then
aspirate the liquid. Repeat these steps at least six times. After washing, the plate is
inverted and tapped dry on absorbent tissue paper.
Alternatively, the Wash buffer may be put into a squirt bottle. If a squirt bottle is used,
empty the wells by a firm ‘shake-out’ action and flood the plate with Wash buffer,
completely filling all wells. Repeat these steps at least six times. After washing, the plate
is inverted and tapped dry on absorbent tissue paper.

Note: When you have too much airbubbles by using a squirt bottle, you can replace wash
buffer by PBS during the last washing step.

• When using an automated washing device, follow operating instructions carefully.

Data analysis
In each experiment a standard curve should be run, consisting of 7 standard dilutions, from
which a concentration-response relationship is generated. Construct the standard curve by
plotting the mean OD of each standard dilutions (Std 1-7) minus the mean Blank (see formula
below) on the y-axis against the corresponding concentration on the x-axis.

Formula: OD = mean ODstd 1-7/Sample - mean ODblank

Most laboratories have (plate reader) software that allows various methods of curve fitting.
Since ELISA data are essentially sigmoid rather than linear, we recommend using the 4- or
5-parameter logistic fit for quantitative analysis of the samples. Alternatively, a linear
regression curve may be acceptable for the linear portion of the curve consisting of at least 3
concentrations.
After selection of the regression model, the analyte concentration in unknown samples can
be interpolated from the standard curve. The OD value of the sample should fall within the
standard curve. Samples showing an OD below the lowest concentration of the standard curve
should be re-analyzed at a lower dilution. Samples showing an OD that exceeds the highest
concentration of the standard curve should be re-analyzed at a higher dilution.
If samples have been diluted, the calculated concentration must be multiplied by the dilution
factor.

13
Assay procedure
All solutions should be at RT prior to use.

1. Add 50 µl of diluted coating antibody solution to each well of the ELISA plate and fill up
to 100 µl with PBS. Seal the plate to prevent evaporation.

2. Incubate overnight at 4 ºC (or alternatively 2 hours at 37 ºC).

3. Remove coating antibody solution and wash the wells at least six times with Wash buffer.

4. Add 200 µl of Blocking buffer to each well.

5. Seal the plate and incubate for 1 hour at 37 ºC.

6. Remove the Blocking buffer (do not wash the wells).

7. Add 100 µl of diluted standard/blank/samples to each well.

8. Seal the plate and incubate for 2 hours at 37 ºC (or alternatively overnight at 4 ºC).

9. Remove standards/samples and wash the wells at least six times with Wash buffer.

10. Add 100 µl of diluted detection antibody solution to each well.

11. Seal the plate and incubate for 1 hour at 37 ºC.

12. Remove detection antibody solution and wash the wells at least six times with Wash buffer.

13. Add 100 µl of diluted SPP conjugate to each well.

14. Seal the plate and incubate for 1 hour at 37 ºC.

15. Remove SPP conjugate and wash the wells at least six times with Wash buffer.

16. Add 100 µl of TMB substrate solution into each well.

17. Leave the plate for 20 min at RT in the dark.

Note: The substrate produces a soluble blue end product that can be read at 370 or 655 nm.

18. After substrate incubation, do not empty the wells. Stop the reaction by adding 100 µl
of Stop solution (resulting in a yellow solution) and read the plate at 450 nm within
30 minutes.

14
Troubleshooting

Problem Possible cause Solution

-- Ensure accurate pipetting of volume and avoid

Inaccurate pipetting air bubbles.
-- Check pipettes.
Inadequate mixing of reagents -- Mix reagents adequately.
Poor consistency of -- Increase the stringency of washes (particularly
replicates Inadequate washing after the ‘detection antibody’ incubation step).
-- Check function of the plate washer.
Evaporation of solutions -- Ensure precise sealing of the plate.
Non-homogenous samples or -- Mix samples thoroughly and remove particulates
with high particulate matter by centrifugation.
Incubation time of TMB
-- Shorter incubation time of TMB substrate.
substrate solution is too long
Incubation temperature of TMB -- Decrease incubation temperature of TMB
substrate solution is too high substrate.
ODblank values higher
than 0.3 Working solutions were -- Solutions should be clear and colorless. Use a
contaminated clean container before addition into wells.
Detection antibody or conjugate
-- Ensure proper dilution of detection antibody or
dilution was too concentrated or
conjugate and incubation time.
left too long on the plate
Improper storage of -- Avoid prolonged exposure to light and heat.
reconstituted SPP -- Avoid storage at RT.
Incorrect incubation times or -- Ensure sufficient incubation times.
temperature -- Reagent solutions should be at RT before use.
Improper quality or pH of -- Use distilled water, and not tap water.
distilled water -- Check quality and pH of distilled water.
No signal or low OD Improper antibody, SPP or -- Ensure proper dilution of antibody, SPP and
values for standards standard dilution standard.
Degradation of antibodies or SPP -- Follow recommended storage conditions.
Overly high washing / aspiration
-- Check function of washing system or utilize
pressure from automated plate
manual washing.
washer.
Working solutions contain -- Avoid adding sodium azide in solutions, as this is
sodium azide a ‘peroxidase activity’ inhibitor.
-- Ensure proper dilution of standards
Poor standard curve Improper standard dilutions
(follow ‘two-fold dilutions’ guidelines).
(linearity and
dynamic range) -- Ensure accurate pipetting of volume.
Inaccurate pipetting
-- Check pipettes.

More information can be found on: www.ucytech.com/FAQs_elisa

15
 Technical assistance
If you require assistance, information or have any questions, please contact our company:
U-CyTech biosciences
Phone: +31.30.253 5960
E-mail: [email protected]

On our website (www.ucytech.com/manuals) you can find: Manuals, Typical data and
MSDS of our ELISA kits.

ELISA kit versie 171121