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Cytokine ELISA
Introduction
Due to the amplifying potential of enzyme labels, immunoassays which utilize
enzyme-conjugated antibodies have become increasingly popular because of their
high specificity and sensitivity.1 In 1971, Engvall and Perlmann2 coined the term
“enzyme-linked immunosorbent assay” which is perhaps better known by the
acronym, “ELISA”, to describe an enzyme-based immunoassay method which is
useful for measuring antigen concentrations.

Cytokine sandwich ELISA are sensitive enzyme immunoassays that can specifically
detect and quantitate the concentration of soluble cytokine and chemokine
proteins. The basic cytokine sandwich ELISA method makes use of highly-purified
anti-cytokine antibodies (capture antibodies) which are noncovalently adsorbed
(“coated” – primarily as a result of hydrophobic interactions) onto plastic
microwell plates. After plate washings, the immobilized antibodies serve to
specifically capture soluble cytokine proteins present in samples which were
applied to the plate. After washing away unbound material, the captured
cytokine proteins are detected by biotin-conjugated anti-cytokine antibodies
(detection antibodies) followed by an enzyme-labeled avidin or streptavidin
stage. Following the addition of a chromogenic substrate-containing solution,
the level of coloured product generated by the bound, enzyme-linked detection
reagents can be conveniently measured spectrophotometrically using an ELISA-
plate reader at an appropriate optical density (OD). Data storage and reanalysis
are greatly simplified when the plate reader is connected to a computer.

By including serial dilutions of a standard cytokine protein solution of known

concentration, the sandwich ELISA supports the development of standard curves.
Standard curves (aka“calibration curves”) are generally plotted as the standard
cytokine protein concentration (typically ng or pg of cytokine/ml) versus the
corresponding mean OD value of replicates. The concentrations of the putative
cytokine-containing samples can be interpolated from the standard curve. This
process is made easier by using an ELISA computer software program.3 Generally,
it is useful to perform a dilution series of the unknown samples to be assured that
the OD will fall within the linear portion of the standard curve. Depending on
the nature of the ELISA reagents used, investigators may choose to apply
different curve fit analysis to their data, including either linear-log, log-log, or
four-parameter transformations.1,4,5
Although opinions differ, one convention for determining the ELISA sensitivity is
to choose the lowest cytokine concentration that gives a signal which is at least
two or three standard deviations above the mean background signal value.6,7
Because of the enzyme-mediated amplification of the detection antibody signal,
the sandwich ELISA can measure physiologically relevant (i.e., > 5-10 pg/ml)
concentrations of specific cytokine and chemokine proteins, which are present in
mixed cytokine milieus, e.g., from stimulated lymphocyte culture supernatants.
Although many different types of enzymes have been used, horseradish
peroxidase (HRP) and alkaline phosphatase (AKP) are the enzymes that are often
employed in ELISA methods.1,8

Application Notes
Cytokine sandwich ELISA are exquisitely specific because antibodies directed
against two or more distinct epitopes are required.9 Therefore, sandwich ELISA
can discriminate between cytokines that can have overlapping biological
functions which are not resolvable in a bioassay. Although cytokine sandwich
ELISA are very useful for cytokine detection and measurement, several limitations
for the interpretation of ELISA data must be mentioned.9 For example, because
test samples often come from tissue culture supernatants or biological fluids
which are conditioned with cytokines produced by mixed cell populations, the
ELISA data does not provide direct information on the identities and frequencies
of individual cytokine producing cells. Techniques such as the
"Immunofluorescent Staining of Intracellular Cytokines" are required for this
latter type of analysis.

Several key issues need to be considered when designing experiments that involve
cytokine and chemokine protein measurements using sandwich ELISA. For
instance, it is well known that cytokine production by stimulated cell populations
is transient and that the kinetics of expression of different cytokine genes can
vary. For these reasons, it may therefore be necessary to collect test samples at
several time points to better characterize cytokine-production by an experimental
animal or by a cultured cell population. As an example, in the case of stimulated
mouse CD4+ T cell populations, the levels of IL-2 produced are detected relatively
early after stimulation whereas the accumulated levels of IL-5 protein rise later in
culture.10

It should also be noted that cytokine production can be stimulus- and cell subset-
dependent. For example, in the case of T cells, it is well known that naive T cells
have a limited cytokine production capability (i.e., primarily can produce IL-2)
whereas memory T cells can produce high levels and different types of cytokine
proteins including IFN-g and IL-4, as well as IL-2. 11,12 Moreover, T cell subsets
have been found to produce cytokines differentially in response to different
stimuli.12,13 Another consideration is that cytokine protein concentrations,
measured at any one time point, may reflect the concurrent processes of cytokine
secretion, cytokine uptake by cells and cytokine protein degradation. Because of
these processes, the measured level of cytokine protein may significantly
underestimate the actual cytokine-producing potential of cells. In these cases, it
may be necessary to use complementary techniques such as multi-probe
ribonuclease protection assay analysis or immunofluorescent intracellular cytokine
staining with flow cytometric analysis to gauge the relative levels of cytokine
expression by various test cell populations.

The levels of immunoreactive cytokine proteins detected by ELISA may or may not
correlate directly with the levels of bioactive cytokine protein.9,14 For example, an
ELISA may utilize anti-cytokine antibodies that cannot discriminate between the
precursor (inactive) and mature (bioactive) forms of a cytokine protein such as
TGFb1. Moreover, an ELISA may detect partially-degraded cytokine proteins
which have retained their immunoreactive properties (i.e., at least two
recognizable epitopes) but may have lost their bioactivity. In conclusion, cytokine
sandwich ELISA are useful indicators of the presence and levels of cytokine and
chemokine proteins but they do not actually provide information concerning the
biological potency of the detected proteins.

With these caveats in mind, one can infer from the presence and amount of
cytokine protein detected, the potential mechanisms by which particular effector
cell populations perform their functions. Moreover, sandwich ELISAs can detect
soluble cytokine receptors which may be important for cytokine regulation.
Soluble cytokine receptors may act as antagonists or as carrier proteins in vivo and
may serve as disease markers in in vitro tests.15 It should be noted that in addition
to providing a rich source of information for clinical and basic science research
studies, sandwich ELISA for measuring cytokines and their receptors have become
increasingly important as diagnostic tools and for monitoring therapeutic
regimens,16 e.g., biological response modification regimens utilizing recombinant
cytokine proteins. In the latter cases, highly optimized sandwich ELISA kits
designed to minimize interference or nonspecific reactivities presented by patient
samples is highly desirable.

ELISA Protocol General Procedure

Capture antibody:
1. Dilute the purified anti-cytokine capture antibody to 1-4 µg/mla in Binding
Solution. Add 50 µl of diluted antibody to the wells of an enhanced protein-
binding ELISA plate (e.g.,Nunc Maxisorb; cat. no. 446469).

2. Seal plate to prevent evaporation. Incubate overnight at 4°C.

Blocking:
3. Bring the plate to RT, remove the capture antibody solution, and block non-
specific binding by adding 200 µl of Blocking Buffer per well.

4. Seal plate and incubate at RT for 1-2 hr.

5. Wash ≥3 times d with PBS/Tween ® .

Standards and Samples:

6. Add standardsb,c and samples (diluted in Blocking Buffer/Tween ®h at 100 µl per
well.

7. Seal the plate and incubate it for 2-4 hr at RT or overnight at 4°C.e

8. Wash ≥4 timesd with PBS/Tween® .

Detection antibody:
9. Dilute the biotinylated anti-cytokine detection antibody to 0.5-2 µg/ml in
Blocking Buffer/Tween®.h Add 100 µl of diluted antibody to each well.

10. Seal the plate and incubate it for 1 hr at RT.

11. Wash ≥4 timesd with PBS/Tween® .

Avidin-Horseradish Peroxidase (Av-HRP):
12. Dilute the Av-HRP conjugate (cat. no. 13007E) or other enzyme conjugatec,f to
its pre-titered optimal concen-tration in Blocking Buffer/Tween®. Add 100 µl
per well.

13. Seal the plate and incubate it at RT for 30 min.

14. Wash ≥5 times d with PBS/Tween® .

Substrate:
15. Thaw ABTS Substrate Solutionc,f within 20 min of use. Add 100 µl of 3% H2O2
per 11 ml of substrate and vor-tex. Immediately dispense 100 µl into each
well. Incubate at RT (5-80 min) for colour development. The colour reaction
can be stopped by adding 50 µl of Stopping Solution.

16. Read the optical density (OD) for each well with a microplate reader set to 405
nm.g

SOLUTIONS:
§ Binding Solution: 0.1 M Na2HPO4 , adjust pH to 9.0 with 0.1 M NaH2PO4.
§ PBS Solution: 80.0 g NaCl, 11.6 g Na2HPO4 , 2.0 g KH2PO4 , 2.0 g KCl; q.s. to 10
L; pH to 7.0.
§ PBS/Tween®: 0.5 ml of Tween® -20 in 1 L PBS.
§ Blocking Buffer: Prepare 10% fetal bovine serum (FBS), 10% newborn calf
serum (NBCS ) or 1% BSA (immunoassay grade) in PBS. The Blocking Buffer
should be filtered to remove particulates before use.
§ Blocking Buffer/Tween®: Add 0.5 ml Tween® -20 to 1 L Blocking Buffer.
§ ABTS Substrate Solution: Add 150 mg 2,2’-Azino-bis-(3-ethylbenzthiazoline-6-
sulfonic acid) (e.g., Sigma, cat. no. A-1888) to 500 ml of 0.1 M anhydrous citric
acid (e.g., Fisher; cat. no. A-940) in dd H20; pH to 4.35 with NaOH. Aliquot 11
ml per vial and store at -20°C. Add 100 µl 3% H2O2 prior to use.
§ 3% H2O2 Solution: Add 10 ml of 30% H2O2 to 90 ml of H2O2. Protect from
prolonged exposure to light.
§ Stopping Solution: (20% SDS/50% DMF): Add 50 ml of dimethylformamide
(DMF) (Pierce, cat. no. 20672) to 50 ml dd H20, then add 20.0 g sodium dodecyl
sulfate (SDS) (CMS, cat. no. 424-749).

Cytokine ELISA Helpful Hints

a. To determine the optimal signal and lowest background for the ELISA, the
capture antibody (1-4 µg/ml) and detection antibody (0.25-2 µg/ml should be
titrated against each other in a preliminary experiment. An appropriate range
of serial dilutions for the cytokine standard should be included. A suggested
range is generally provided on the Technical Data Sheet (TDS) for ELISA
reagents. Generally, use of the capture antibody at 2 µg/ml and the detecting
antibody at 1 µg/ml provides strong ELISA signals with low back-ground.
b. CYTOKINE STANDARD HANDLING: Please read the TDS for each recombinant
cytokine carefully. Handling instructions are lot-specific. For maximum
recovery of cytokine, the vial of cytokine should be quick-spun before
opening. Lyophilized cytokines should be reconstituted as indicated in the lot-
specific TDS. BD Biosciences recommends keeping the cytokine solution in a
concentrated form (e.g., ≥1 µg/ml) and in the presence of a protein carrier for
long-term storage.
c. The linear region of cytokine ELISA standard curves are generally obtainable in
a series of eight two-fold dilutions of the cytokine standard, from 2000 pg/ml
to 15 pg/ml. To increase sensitivity beyond that obtainable with the standard
 ELISA protocol, amplification kits, tertiary reagents, or alternate
enzyme/substrate systems can be used.
d. High backgrounds in blank wells (i.e., OD > 0.20) or poor consistency of
replicates can be overcome by increasing the stringency of washes and
optimizing the concentration of capture and detection antibodies. For
example, during washes, the wells can be soaked for ~ 1 minute intervals.
Moreover, lower concentrations of detecting antibody or more washes after
the detecting antibody stage can reduce background.
e. For optimal sensitivity, overnight incubation of standards and samples
is recommended.
f. If using peroxidase as the enzyme for colour development, avoid sodium azide
in wash buffers and diluents, as this is an inhibitor of peroxidase activity.
g. If no signal is observed, check the following: a) verify that appropriate
antibody clones were used; b) check the activity of the enzyme/substrate
system: e.g.,coat 1 µg/ml of biotinylated detecting antibody in several wells in
binding buffer for a few hr. After blocking, wash several times then proceed
with the cytokine ELISA protocol from Step 13. If the enzyme/substrate system
is active, then a strong signal should be seen; c) verify the activity of cytokine
standard or try a new sample of standard.
h. When measuring cytokines in complex fluids, such as serum, sample
diluents which include irrelevant Ig are suggested.17

References:
1. Crowther, J. R. 1995. ELISA. Theory and Practice. Methods Mol. Biol. 42:1-223.
2. Engvall, E., and P. Perlmann. 1971. Enzyme-linked immunosorbent assay (ELISA). Quantitative
assay of immunoglobulin G. I mmunochem.8:871-874.
3. Davies, C. 1994. Principles. In The Immunoassay Handbook. D. Wild, ed. Stockton Press, New
York, p. 3-47.
4. Rogers, R. P. C. 1984. Data Analysis and Quality Control of Assays: A Practical Primer. In
Practical Immuno Assay. W. R. Butt, ed. Marcel Dekker, Inc., New York.
5. Davies, C. 1994. Calibration curve fitting. In The Immunoassay Handbook. D. Wild, ed, New
York, p. 118-123.
6. Davies, C. 1994. Concepts. In The Immunoassay Handbook. D. Wild, ed. Stockton Press, New
York, p. 83-115.
7. Pathak, S. S., A. van Oudenaren, and H. F. J. Savelkoul. 1997. Quantification of
immunoglobulin concentration by ELISA. In Immunology Methods Manual, vol. 2. I. Lefkovitz,
ed. Academic Press Inc., San Diego, p. 1056-1075.
8. Wild, D., and C. Davies. 1994. Components. In The Immunoassay Handbook. D. Wild, ed. Press,
New York, p. 49-82.
9. Mosmann, T. R., and T. A. T. Fong. 1989. Specific assays for cytokine production by T cells. J.
Immunol. Meth.116:151-158.
10. Hobbs, M. V., W. O. Weigle, D. J. Noonan, B. E. Torbett, R. J. McEvilly, R. J. Koch, G. J. Cardenas,
and D. N. Ernst. 1993. Patterns of cytokine gene expression by CD4 + T cells from young and
old mice. J. Immunol. 150:3602-3614.
11. Ehlers, S., and K. A. Smith. 1991. Differentiation of T cell lymphokine gene expression: The in
vitro acquisition of T cell memory. J. Exp. Med.173:25-36.
12. Cerottini, J.C., and H. R. MacDonald. 1989. The cellular basis of T-cell memory. Annu. Rev.
Immunol. 7:77-89.
13. Farber, D. L., M. Luqman, O. Acuto, and K. Bottomly. 1995. Control of memory CD4 T cell
activation: MHC class II molecules on APCs and CD4 ligation inhibit memory but not naive CD4
T cells. Immunity 2:249-259.
14. Carter, L. L., and S. L. Swain. 1997. Single cell analyses of cytokine production. Curr. Opin.
Immunol.9:177-182.
15. Callard, R. E., and A. J. H. Gearing. 1994. Cytokine receptor superfamilies. In The Cytokine Facts
Book. Academic Press Inc., San Diego, p. 18-27.
16. Rossio, J. L. 1997. Cytokines and immune cell products. In Weir's Handbook of Experimental
Immunology. Fifth Edition. D. M. Weir, L. A. Herzenberg, L. A. Herzenberg, and C. Blackwell,
eds. Blackwell Science, Inc., Cambridge, MA.
17. Abrams, J.S. 1995. Immunoenzymetric assay of mouse and human cytokines using NIP-labeled
anti-cytokine antibodies. Current Protocols in Immunology (J. Coligan, A. Kruisbeek, D.
Margulies, E. Shevach, W. Strober, eds). John Wiley and Sons, New York. Unit 6.20.