Measurement of Cytokine Levels

Cytokine levels can be measured in a number of bodily fluids, including serum, plasma, urine, supernatants, mucus, and saliva. A range of techniques is available to measure cytokine levels, including enzyme-linked immuno sorbent assay (ELISA), multiplex technology, ELISPOT and flow cytometry.  Below are basic descriptions of some of these methods.

Enzyme Linked Immuno Sorbent Assay (ELISA)
What will the assay tell us?
The ELISA is the most commonly used immunoassay to quantify cytokine levels. These assays
are available in the form of commercial kits and are widely used because of their specificity,
sensitivity, ease, and convenience.
For further details regarding this general method, see “Common Immune Measures.”
How are samples collected and stored?
For plasma samples, whole blood should be collected in sterile tubes with anticoagulants, placed
on ice immediately, and spun down as soon as possible, preferable within 30 minutes. Many
cytokines have a short half-life and begin to degrade once drawn. Small Aliquots (e.g., .5ml) of
plasma are pipetted into freezer tubes and frozen in an ultra-cold freezer. For serum samples, no
anti-coagulant is used and samples are allowed to clot at room temperature for 30 minutes before
spinning. It is best to avoid repeated freeze-thawing of samples. 
What type of ELISA should I use?
Many cytokine ELISA kits come in high and low sensitivity versions. These differ in the range of
detectable concentrations, with high sensitivity kits reliably detecting lower concentrations of
cytokines. hen available, these kits are better able to detect levels of cytokines in unstimulated
samples than the low sensitivity kits.
What are the advantages and disadvantages of an ELISA?
High specificity, reliability, and the relatively low cost and ease of use make the ELISA a good
option for assessing cytokine levels. ELISA kits permit the assessment of 38 samples run in
duplicate and cost around $400-$600 per kit. One disadvantage of this method is the possibility
of capturing biologically inactive cytokines and their fragments.  Another issue is the detection
range of the ELISA. Often cytokines are present at levels too low for detection in healthy individuals.
Thus, it is important to examine whether the cytokine falls within the standard curve for the
specific ELISA and thus is present at levels that are reliably detectable. The ‘coefficient of
variability’ (CV) provides information on the reproducibility of an assay and should ideally
be < 5% for the intra-assay (between duplicates tested within assay) CV and < 10% for the
inter-assay (between assay) CV. 
Factors to consider in measuring cytokine levels
A number of factors have been demonstrated to be associated with cytokines levels, including age,
sex, and race. Many cytokines also have a clear circadian rhythm, so samples should be drawn at
the same time of day for comparisons across individuals. Other heath factors that have been
related to cytokine levels include smoking, alcohol use, physical activity, genetics, current acute
or chronic illness, and medication use. Findings differ depending on the cytokine of interest, but
these factors should be taken into consideration in interpreting findings.

Assays for Cytokine Production
In addition to measuring levels of cytokines in samples, whole blood or isolated cell subtypes
(e.g., peripheral blood mononuclear cells (PMBCs)) can be stimulated to produce cytokines in
vitro. It has been suggested that measures of cytokine production provide a more biologically
meaningful measure of cytokines than plasma levels (Whiteside, 1994). Stimulated cytokine
production has been demonstrated to be a stable trait among healthy volunteers (e.g., Friberg
et al, 1994), making it a possible risk factor in susceptibility to immune-related disease. 
What will the assay tell us?
Spontaneous production of cytokines by cells cultured in the laboratory provides a measure of in
vivo activation of these cells. Stimulated production of cytokines by cells exposed to antigens in
the laboratory provides a measure of immune potential or competence. More than one cytokine is
released in response to stimulation, making it possible to examine cytokine arrays. It is also
possible to examine the kinetics of cytokine responses to stimulation, by incubating the culture
for varying periods. 
How does the assay work?
To examine spontaneous production of cytokines, whole blood or isolated cells are incubated for
a period in medium and then the culture supernatant is collected and frozen for future determination
of cytokine levels using ELISA. The cells for this assay can be harvested using centrifugation with
a density separating agent such as ficoll to isolate peripheral blood mononuclear cells, or using
flow cytometry or magnetic beads to isolate a specific type of lymphocyte (e.g., a T cell or B cell).
To assay stimulated cytokine production, whole blood or isolated cells are cultured in medium in
the presence of a stimulant. The specific stimulant depends on the immune pathway of interest.
For example, lipopolysaccharide (LPS), a component of the cell membrane of gram negative
bacteria, is frequently used to stimulate the activation of monocytes/macrophages and thus to
stimulate the release of pro-inflammatory cytokines. In contrast phytohemagluttinin (PHA) is
predominantly a stimulant of T cells. Following incubation with the stimulant, the culture is spun
down and the supernatant harvested and stored in an ultra-low freeze for future assay in batches.
Levels of cytokines in culture supernatants can be determined using ELISA or multiplex assay.

Multiplex Bead Assay
What is a multiplex bead assay?
Stimulation of blood or isolated cells results in the release of a cascade of biologically-related
cytokines. For example, IL-6, TNF-alpha, and IL-1beta are involved in the inflammatory cascade,
IL-2, IFN-gamma, and TNF-alpha characterize Th-1 cell activation, and IL-4, IL-5, and IL-10 are
associated with a Th2 type response. 
A multiplex bead immunoassay permits the simultaneous measurement of an array of cytokines
in a single, small volume sample. Using this method, it is possible to profile cytokine responses.
The assay measures levels if a number of different cytokines in a single sample.
How does the assay work?
In this assay, the sample is cultured with distinct fluorescently labeled beads, coated with a
cytokine-specific antibody. Beads specific for the different cytokines of interest are placed in a
single tube with the sample. The capture antibody binds to the cytokine. Biotin-conjugated detection
antibody specific for the cytokine of interest is then added, followed by fluorescein- labeled
streptavidin. The level of fluorescence bound to the beads is quantified using the Luminex platform.
This is a flow analysis system designed to simultaneously measure multiple analytes.
Kits are commercially available and it is possible to select panels of cytokines (e.g., inflammatory
cytokines) or to customize one’s own panel
What are the advantages and disadvantages of multiplex bead assays?
This technology makes it possible to detect the levels of many cytokines (>10) in a single 50 ul
sample. Thus, this technique is cost effective, saves time and offers flexibility. Although it offers a
great system for assessing stimulated levels of cytokines, the detection range is not sensitive to
circulating levels of many cytokines in healthy individuals, which often fall below the lower limit of
detection. In these instances, it is possible that a high sensitivity ELISA may offer greater sensitivity.
As with ELISAs, the investigator must explore whether expected levels will fall within the standard
range of the kit. To achieve this, stimulated levels samples will often need to be diluted prior to
assay. This may be difficulty when cytokines are present at very different levels within a single
sample.

ELISPOT
What will the assay tell us?
ELISPOTs provide a different assay for evaluating cell activation following stimulation with an
antigen. This assay is used to determine the frequency of cells in a sample that are capable of
releasing cytokines following stimulation.
How does the assay work?
The ELISPOT is an enzyme linked-immune assay that is similar to ELISA and measures immune
cell secreted cytokine levels in response to in vitro stimulation with an antigen within the well of a
plate coated with antibodies specific for the cytokine of interest.
How are samples prepared?
For this assay, cells are isolated from whole blood. This can be performed using centrifugation
with a density separating agent such as ficoll to isolate peripheral blood mononuclear cells, or
using flow cytometry or magnetic beads to isolate a specific type of lymphocyte (e.g., a T cell or
B cell). The number of isolated cells in the sample has to be determined before cells are added to
the ELISPOT plate. 
How is the assay conducted?
The isolated cells can be stimulated with a specific antigen or mitogen to produce cytokine either
before they are added to the ELISPOT wells, or directly while on the plate.  Stimulated cells are
added to a plate which is coated with capture antibodies. The plate is then incubated for a specific
time at a particular temperature, depending on the cytokine examined. During incubation, the cells
react with the capture antibodies on the plate and producing a spot or ring of peptide-antibody
complexes that reflects both the kinetics and quality of cytokine production by individual cells
during the test period. At the end of incubation, the plate is washed, and an enzyme-linked
antibody is added that binds to the cytokine. Chromogenic substrate is then added that depicts
the cytokine producing cells as colored points.
How are results interpreted?
The number of colored spots produced in the assay reflects the amount of cytokine produced by
the cells. Spots are examined for size, density, and morphology to determine kinetics and type of
cells secreting the cytokine. For example, small, dense spots reflect the slow stable release of
cytokines. In contrast, larger, more scattered spots reflect faster release. Semi-automated and
automated methods exist for reading ELISPOT data, using a plate reader and software programs.
The researcher is required to define counting parameters, such as spot size. Data is usually
reported as number of spots per a specific number of cells.
What are the advantages and disadvantages of ELISPOT?
ELISPOT offers information about the frequency of specific cells that are activated and producing
cytokines in a sample. As with ELISA, a limitation is the possibility of detecting non-biologically
active cytokines. They provide a more specific measure of immune function than ELISAs. However,
ELISPOT assays are substantially more expensive than ELISAs.

Flow Cytometry Techniques for the Measurement of Extracellular and Intracellular Cytokines
What will the assay tell us?  
Flow cytometry can be used to assess cytokines levels in both blood and within single cells.
How does the assay work?
To measure intracellular cytokines, cells are treated so that their cell membranes become
permeable, allowing antibody to reach the cytokine inside the cell. This enables the staining of
intracellular cytokines. In this technique, cells are treated with a protein transport inhibitor, then
fixed and one of a number of methods is used to make the cell more permeable. The cell is then
stimulated and exposed to labeled anti-cytokine antibodies that bind to the fixed intracellular
cytokines. Flow cytometry is used to quantify cytokines within single cells
(See “Common Immune Measures” for further details regarding flow cytometry).
To measure cytokine secretion by single cells, a number of commercial assays are now available.
Whole blood or isolated cells are first stimulated by incubation with antigen. Cells are then exposed
to a conjugate of anti-CD45 and anti-cytokine antibody. This attaches to the surface of leukocytes
and then “catches” the specific cytokine if it is released by the activated cell. Cells are then
exposed to a cytokine-specific detection antibody labeled with a fluorochrome which attaches to
cytokine-producing cells. The level of fluorochrome is quantified by flow cytometry. 
What are the advantages and disadvantages of these flow cytometry methods?
These methods provide a highly sensitive method for determining antigen-specific, cytokine
secreting cells. Benefits of this approach include quantifying not only the percentage or number
of certain cytokine-producing cells in the sample but also the specific cell type, and the
expression of other related molecules.

References
For a general review of cytokine measurement assays, see the following: 
Banks RE: Measurement of cytokines in clinical samples using immunoassays:
Problems and pitfalls. Crit Rev Clin Lab Sci 37:131-182, 2000.
Friberg D, Bryant J, Shannon W, Whiteside TL: In vitro cytokine production by normal
peripheral blood monouclear cells to measure their immunocompetence and state of
activation. Clin Diagn Lab Immunol 1:261-268, 1994.
Whiteside TL: Cytokine assays. Bio Techniques 33: S4-S15, 2002.
Whiteside TL: Cytokines and cytokine measurements in a clinical laboratory.
Clin Diagn Lab Immunol 1:257-260, 1994.

Core-E MainBiological Measures Used