Cytokine%252520 ELISA%252520 Methods

Table of Contents
I. Introduction....................................................................................................................................2
II. Cytokines
Description and Role in Immunity ..................................................................................................3
Primary Activity and Primary Source ..............................................................................................4
Cytokine Production in Leukocytes ................................................................................................7
Chemokine Activation in Leukocytes ..............................................................................................8
Summary of Cytokine Actions and Classifications ........................................................................10
List of Specific Cytokine Actions ..................................................................................................12
III. Cytokine ELISAs (pre-coated plates)
Background ..................................................................................................................................18
Assay Optimization ......................................................................................................................18
The Importance of Proper Washing ..............................................................................................18
General ELISA Procedure..............................................................................................................19
EASIA™ Kits- Specialty ELISA Kits for Use with Clinical Serum Samples ....................................20
Cross-reactivity of BioSource Human ELISA Kits with non-human Primates ..............................22
IV. Cytokine CytoSet™ Antibody Pairs
Background ..................................................................................................................................24
Typical Standard Curve ................................................................................................................24
Recommended Buffers and Solutions (and alternate choices) ....................................................25
CytoSet Recommended Assay Procedure ....................................................................................25
Plate Coating Tips ........................................................................................................................26
Assay Optimization ......................................................................................................................26
Frequently Asked Questions..........................................................................................................30
V. Methods for ELISA Sample and Tissue Preparation
Human Serum and Plasma Samples ............................................................................................31
Tissue Culture Samples ................................................................................................................32
Tissue Extraction Buffer ................................................................................................................32
Method for Isolating Intracellular and Extracellular Proteins from Whole Tissue ........................34
Cell Extraction Buffer Protocol (for use in ELISA) ........................................................................41
Rat Liver: Sample Preparation ......................................................................................................43
Rat Blood Sample: Cardiac Puncture for Blood Sample in Rats – Open Chest Method................43
Rat Blood Sample: Cardiac Puncture for Blood Sample in Rats – Closed Chest Method ............43
Rat Lung: Perfusion to Flush Non-adherent Cells from the Pulmonary Circulation in Rats ..........44
VI. Appendix
Troubleshooting Guide
Pre-coated ELISA Plates..........................................................................................................45
CytoSet Antibody Pairs............................................................................................................50
ELISA References ........................................................................................................................53
CytoSet Antibody Pair References ................................................................................................54
Acknowledgements ......................................................................................................................55
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Introduction
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Cytokines: Description and Role in Immunity
Cytokines are a diverse and unique category of protein mediators that are secreted largely by CD4 positive
T-cells and macrophages. They are produced during the activation and effector phases of innate and specific
immunity. Cytokines play important roles in the development, function and control of the cells of the immune
system as well as many other systems. They are potent molecules that can cause changes in cell proliferation,
differentiation and migration. Like hormones, cytokines initiate action by binding to specific receptors on the
cell surface of target cells. However, unlike hormones, cytokines are usually made by a variety of cells, rather
than in a gland, and act locally (autocrine or paracrine) instead of distally (endocrine). Cytokines may be
pleiotropic (one cytokine, multiple effects), redundant (multiple cytokines, one effect) and antagonistic (one
cytokine inhibits another cytokine). For a good review of immunological roles of cytokines and chemokines, see
Immunology by Janis Kuby, 1994, W.H. Freeman and Co., Cytokine Reference by Joost J. Oppenheim and Marc
Feldman, 2001, Academic Press, or the National Center for Biotechnology (online at www.ncbi.nlm.nih.gov).
Cytokine actions may be grouped into five broad areas:
• Development of cellular and humoral immune responses

• Induction of inflammation
• Regulation of hematopoiesis
• Control of cellular proliferation and differentiation
• Induction of wound healing
Predicting the action(s) of a cytokine is difficult since a cytokine is not alone in the host system. There may
be synergistic or antagonistic actions between different cytokines to produce an unexpected event and/or a
cytokine may start a cascade of cytokine production in which the later cytokines affect the earlier cytokine.
Cytokines do not have "antigen specificity". However, since cytokine receptors are generally expressed only
after a cell is activated by antigen, the resulting immune response is antigen specific.
Secreted primarily from leukocytes, cytokines stimulate the humoral and cellular immune responses, as well
as the activation of phagocytic cells. Cytokines can also be expressed on the cell membrane while others may
be stored in extracellular matrix. Cytokines are typically produced during a short period of time after cell acti-
vation in the context of immune reactions or pathological or inflammatory processes.
Originally, the cytokines were named according to their function (e.g., T-cell growth factor, now called IL-2),
but with the pleiotropy of cytokines observed, this made functional specific names confusing. Historically,
cytokines have been ordered into five different protein families. Main classes of cytokines include:
• Interleukins – Regulate interactions between leukocytes

• Interferons – Response to viral infections (α, β, γ, ω)
• Chemokines – Chemotaxis and inflammation (e.g., IL-8)
• Colony Stimulating Factors – Allow cells to grow (e.g., GM-CSF)
• Tumor Necrosis Factors – Derived from macrophages, anti-tumor activity
Interleukins are cytokines that are secreted from leukocytes. Cytokines that are secreted from lymphocytes
are also termed lymphokines, while those secreted by monocytes or macrophages are termed monokines.
The term interleukin derives from the fact that these cytokines are secreted from leukocytes, but also affect
the cellular responses of leukocytes. More specifically, interleukins are growth factors targeted to cells of
hematopoietic origin.

Interferons are a family of cytokines initially thought to be involved in antiviral responses. However, they are
also potent immunomodulators. They can promote or inhibit the synthesis of antibodies by activated B-cells
and also activate macrophages, natural killer cells, and T-cells. They also possess direct antiproliferative
activities and are cytostatic or cytotoxic for a number of different tumor types.
Chemokines are a family of pro-inflammatory activation inducible cytokines. These proteins are mainly
chemotactic for various cell types. The chemokine name is derived from (chemo)tactic cyto(kine).
Chemokines are multipotent cytokines that localize and enhance inflammation by induced chemotaxis and
cell activation of inflammatory cells at sites of inflammation. Chemokines are also essential mediators of nor-
mal leukocyte trafficking.
Colony Stimulating Factors (CSFs) are cytokines that stimulate the proliferation of specific pluripotent stem
cells of the bone marrow in adults. Granulocyte-CSF (G-CSF) is specific for the proliferation and differentia-
tion of hematopoietic progenitor cells committed to the granulocyte lineage. Macrophage-CSF (M-CSF) influ-
ences the proliferation and differentiation of stem cells into macrophages but mainly the growth and differ-
entiation of monocytes. Granulocyte-macrophage-CSF (GM-CSF) is responsible for the growth and develop-
ment of granulocytes and macrophage progenitor cells.
The Tumor Necrosis Factor (TNF) family consists of two molecular species, TNF-α and TNF-β. TNF-α
induces expression of other autocrine growth factors, increases cellular responsiveness to growth factors
and induces signaling pathways that lead to proliferation. TNF-α also induces expression of a number of
nuclear proto-oncogenes as well as other interleukins. TNF-β is characterized by its ability to kill a number
of different cell types as well as the ability to induce terminal differentiation in others. The induction of
TNF-β results from elevations of IL-2 as well as the interaction of antigen with T-cell receptors.
Table 1
A limited sample of cytokines listed with their primary activity and primary source.
Interleukin Primary Activity Principal Source

IL-1-α & -β Enhance the activation of T-cells in Macrophages and other antigen
response to antigen presenting cells (APCs)
IL-2 Proliferation of B-cells and activated Activated TH1 cells, NK cells
T-cells, NK function
IL-3 Growth of hematopoietic progenitor cells Activated T-cells
IL-4 B-cell proliferation, eosinophil and mast cell growth TH2 cells and mast cells
and function, IgE and class II MHC expression on
B-cells, inhibition of monokine production
IL-5 Eosinophil growth and function TH2 cells and mast cells
IL-6 Acute phase response, B-cell proliferation, Activated TH2 cells, APCs, and
thrombopoiesis, synergistic with IL-1 and TNF other somatic cells
on T-cells
IL-7 T and B lymphopoiesis Thymic and marrow stromal cells

IL-8 Chemoattractant for neutrophils and T-cells Macrophages, other somatic cells
IL-9 Hematopoietic and thymopoietic effects T-cells
IL-10 Inhibits cytokine production, promotes B-cell Activated TH2 cells, CD8+ T- and
proliferation and antibody production, suppress B-cells, macrophages
cellular immunity, mast cell growth

IL-11 Synergistic hematopoietic and thrombopoietic effects Stromal cells
IL-12 Proliferation of NK cells, IFN-γ production, B-cells, macrophages
promotes cell-mediated immune functions
IL-13 IL-4-like activity TH2 cells
IL-14 Induces B-cell proliferation, inhibits Ig secretion, T-cells
selectively expands some B-cell populations
IL-15 T-cell growth factor with similar T-cell T-cells
activities to IL-2
IL-16 CD4+ T lymphocyte attractant CD8+ T-cells
IL-17 Stimulate epithelial, endothelial and fibroblastic Activated memory B-cells
cells to secrete IL-6, IL-8, GM-CSF
IL-18 Activates IFN-γ by spleen cells, enhances NK Liver
cell cytotoxicity, augments GM-CSF production
and decreases IL-10 production
IL-19 Drives T-helper cell differentiation towards Keratinocytes and monocytes
a TH2 response
IL-20 Promotes expansion and differentiation of Keratinocytes
keratinocytes
IL-21 Band T-cell proliferation Monocytes
IL-23 T-cell proliferation and IFN-γ production Monocytes
IL-24 Induces proinflammatory cytokines B-cells, CD4+ T-cells, NK cells
IL-26 Unknown (currently under investigation) T-cells, NK cells
IL-27 Anti- and proinflammatory effects Dendritic cells, monocytes
IL-28A Anti-viral responses Dendritic cells, macrophages
IL-29 Anti-viral responses Dendritic cells, macrophages
IL-30 T-cell expansion (TH1 direction) Dendritic cells, macrophages
Interferons Primary Activity Principal Source

IFN-α & -β Antiviral effects, induction of class I MHC on Macrophages, neutrophils and
somatic cells, activation of NK cells and some somatic cells
macrophages
IFN-γ Induces class I MHC on all somatic cells, induces Activated TH1 and NK cells
class II MHC on APCs and somatic cells, activates
macrophages, neutrophils, NK cells, promotes cell-
mediated immunity, antiviral effects

Colony Primary Activity Principal Source
Stimulating Factors
G-CSF Stimulate proliferation of bone marrow stem Activated monocytes,
cells of granulocyte lineage macrophages and neutrophils
M-CSF Stimulate proliferation of bone marrow cells of Monocytes, granulocytes,
macrophage lineage endothelial cells, fibroblasts
GM-CSF Stimulate proliferation of bone marrow stem cells Activated T-cells and
of both granulocyte and macrophage lineage macrophages
Tumor Primary Activity Principal Source

Necrosis Factors
TNF-α Induce expression of other autocrine growth Activated macrophages
factors, of nuclear proto-oncogenes, and induces
signaling pathways that lead to proliferation
TNF-β Kill or induce terminal differentiation of various Cytotoxic T-cells
cell types

Cytokine Production in Leukocytes
For your convenience, chemokine and cytokine activation in specific lymphocytes in shown here and on the
following page.

Chemokine Activation in Leukocytes

Chemokine Activation in Lymphocytes

Cytokine Actions and Classification
As described above, there are many cytokines and these cytokines can have multiple actions. But, for sim-
plicity sake, it is easiest to classify these molecules according to their general actions. Four broad groups
can be identified:
A. Proinflammatory Cytokines (TNF, IL-1, and IL-6)

B. Chemokines (IL-8 and many others)
C. Hematopoiesis
D. Immunomodulatory Cytokines (IL-2, IL-4, IL-5, IFN-γ and others)
A. Proinflammatory Cytokines (TNF, IL-1, and IL-6)

These cytokines are central in helping to initiate and potentiate any inflammatory response, with TNF-α and
IL-1 being major examples. They are secreted by almost any cell and are expressed early in the inflammato-
ry response. The effects include local changes, resulting in mobilization of antigen presenting cells and acti-
vation of endothelial cells to express adhesion molecules. This results in recruitment of inflammatory cells.
IL-1 and TNF also act systemically to induce the acute phase reaction, including fever.
B. Chemokines (IL-8 and many others)

IL-8 is the prototype member of this family but many others exist. These cytokines are important in inflamma-
tory cell recruitment and chemotaxis. Several classes can be recognized due to differences in the amino acid
structure. Different chemokines recruit different classes of inflammatory cells, e.g., neutrophils, lymphocytes or
macrophages. They form a net over endothelial cells and are important in triggering adhesion molecules on the
surface of leukocytes. Chemokines also produce a chemotactic gradient for inflammatory cells to follow.
C. Hematopoiesis
Colony stimulating factors and some interleukins (e.g., IL-3) are crucial for the development of inflammato-
ry cells from the bone marrow precursors. A number of colony stimulating factors are known. However, other
cytokines are also important, and different cytokines are able to induce specific differentiation from the
hemapoietic stem cells and partly committed progenitors.
D. Immunomodulatory Cytokines (IL-2, IL-4, IL-5, IFN-γ and others)

Cytokines are particularly important in the development of T- and B-cell responses. Cytokines are important
both for the activation of lymphocytes and also in determining the type of immune response that develops.
In the initial stage of activation, IL-2 is important. Following the initial stimulation of the T helper lympho-
cytes by the antigen presenting cells, IL-2 is secreted and acts on the cell to activate it. This then leads to
increased IL-2 production, which acts on other helper cells and also on other lymphocytes to activate them.
The type of immune response that develops is also governed by the cytokines that are secreted. Cytokines
secreted by CD4+ T-cells have been divided into two broad groups: T helper 1 (TH1) and T helper 2 (TH2)
types. TH1 cells have a role in cellular immunity, while TH2 cells are involved in humoral immunity. IL-2,
IL-12 and IFN-γ are produced by TH1 cells and are referred to as Type I cytokines, while TH2 cells produce
IL-4, IL-5, IL-10 and IL-13 Type 2 cytokines. It should be noted that there is some overlap, with cytokines
such as IL-3 and TNF-α being included in both groups.

Type I and Type II Cytokines
Often times, researchers classify cytokines (and cells that secrete them) as either TH1 or TH2. TH1 is often
associated with an atopic immune response whereas TH2 cytokines are often associated with a non-atopic
immune response. Below, both TH1 and TH2 cytokies are discussed.
Type I Cytokines
The synthesis and secretion of IL-2 represents the early consequences of antigen or mitogen-induced acti-
vation of mature resting T-cells. IL-2 interaction with its high-affinity receptors promotes clonal expansion of
the effector T-cell population originally activated by antigen.
IL-12 directly induces initial TH1 development and the production of IFN-γ secreting TH1 cells. From a
humoral immunity standpoint, recombinant IL-12 has been shown to be a suppressor of IL-4-induced IgE
production. Also, IL-12 has been hypothesized to play an early role in hematopoiesis.
IFN-γ controls the class of antibody produced in B-cells, up-regulates class I and II MHC complex antigens,
and increases the efficiency of macrophage-mediated killing of intracellular parasites.
Type II Cytokines
IL-4 secretion by TH2 cells elicits humoral response of selective production of IgG, IgE and IgA isotypes. On
B-cells, IL-4 regulates the expression of surface antigens, resulting in the enhancement of the antigen-pre-
senting capacity of B-cells.
IL-5 has a major role in the host as an eosinophil hematopoietic growth factor. IL-5 also can modify basophil
function and induce basophilic differentiation. With effects on both eosinophil and basophil function, IL-5
plays a role in the pathogenic inflammatory responses of hypersensitivity and other diseases associated with
eosinophil infiltration. IL-5 also induces stimulated B-cells to differentiate into Ig-secreting cells.
IL-10 has a broad range of activity on a variety of cell types including both immunosuppressive and
immunostimulatory effects. IL-10 produced in TH2 cells inhibits the production of cytokines, especially IFN-γ
by TH1 cells responding to antigen. Also, IL-10 is a potent down regulator of cell mediated immune response
which results in potent anti-inflammatory activities.
IL-13 is a pleiotropic cytokine produced by activated TH2 cells. In humans, IL-13 brings about changes in
the morphology and phenotypes of monocytes by inducing expression of the IgE receptor and upregulating
expression of class II MHC. Also, IL-13 is involved in IgE switching and the induction of IL-4 independent
IgG4 and IgE synthesis in the presence of T-cells.

List of Specific Cytokine Actions
Granulocyte-macrophage colony stimulating factor (GM-CSF): GM-CSF is a ~14 kDa monomeric protein
with two glycosylation sites. Glycosylation is not required for activity, but the glycosylated form is more
active in vivo. GM-CSF initiates many biological responses. For example, GM-CSF is secreted by T-cells and
macrophages following mitogen or antigen stimulation. GM-CSF is required for the growth and development
of granulocyte and macrophage progenitor cells. In addition, it stimulates myeloblasts and monoblasts and
initiates irreversible differentiation of these cells and induces chemotaxis of eosinophils. Human GM-CSF
activity is measured by the proliferation of the TF-1 cell line and mouse GM-CSF is assayed by measuring the
proliferation of the cell line MC/9.
Interferon-alpha (IFN-α): IFN-α protein size ranges from ~19-26 kDa since there are 23 different variants of
IFN-α. There are two disulfide bonds that are required for full activity. Glycosylation is observed with some iso-
forms. IFN-α is produced by monocytes, lymphoblastoid cells and fibroblasts. IFN-α is a strong antiviral,
antiparasitic, antiproliferative agent. Cell lines to assay the cytopathic activity of IFN-α include MDBK and WISH.
Interferon-beta (IFN-β): IFN-β is a ~20 kDa glycoprotein containing a single disulfide bond that is required
for biological activity. IFN-β is produced by fibroblasts and some endothelial cell types. IFN-β is involved in
humoral immune response regulation and is an antiviral agent. IFN-β is also antiproliferative against a num-
ber of tumor cell lines. The biological activity of IFN-β can be measured by detecting the inhibition of GM-
CSF proliferation of TF-1 cells.
Interferon-gamma (IFN-γ): IFN-γ is a ~16 kDa dimeric protein that contains two cysteines that are not
involved in disulfide bonding. Glycosylation is not required for biological activity. IFN-γ is produced mainly by
T-cells and NK cells activated by antigens and mitogens. IFN-γ is involved in many biological processes. For
example, IFN-γ is antiviral, antiparasitic and inhibits the proliferation of cells. In macrophages IFN-γ induces
the secretion of TNF-α and stimulates the release of reactive oxygen species. It is also involved with bone
growth and inhibits bone resorption. Human IFN-γ activity is measured by detecting the cytostasis of the WiDr
cell line and mouse or rat IFN-γ activity is measured by detecting the cytostasis of the cell line Wehi279.
Interleukin-1 (IL-1): IL-1 is a ~17 kDa protein with no disulfide bonds. There are two distinct molecular forms of
IL-1 (IL-1α and IL-β) derived from two different genes. These two proteins are 26% homologous at the amino
acid level and bind to the same receptor. The predominant function of IL-1 is to enhance the activation of T-cells
in response to antigen and APCs. The IL-1s are secreted primarily by macrophages but also by neutrophils,
endothelial cells, smooth muscle cells and B- and T-cells. IL-1 bioactivity is measured in a proliferation assay
using the cell line D10S. The cell line may be utilized to assay human, swine, rat and mouse IL-1α or IL-1β.
Interleukin-2 (IL-2): IL-2 is a ~15 kDa protein with a single disulfide bond that is critical for activity. IL-2 is
glycosylated but this is not required for biological activity. IL-2 is produced by CD4+ T-cells following acti-
vation by mitogens or allogens. Transformed B-cells, T-cells, Leukemia cells and NK cells also secrete
IL-2. IL-2 causes proliferation of T-cells and is a central regulator of immune responses. There are many cell
lines available to assay the biological activity of IL-2. Mouse CTLL cells are used to determine the prolifera-
tive activity of recombinant IL-2. This cell line will react to human, mouse, swine and rat IL-2.

Interleukin-3 (IL-3): IL-3 is a ~15-17 kDa protein with a single disulfide bond. IL-3 is glycosylated but this
is not required for biological activity. IL-3 is mainly produced by antigen or mitogen activated T-cells. IL-3
provides the cytokine connection between the immune system and the hematopoietic system. For example,
IL-3 supports the proliferation and development of almost all types of hematopoietic progenitor cells and can
act as a chemoattractant for eosinophils. There are many cell lines available to assay the biological activity
of IL-3. Mouse NFS60 cells are used to determine the proliferative activity of recombinant mouse IL-3 and
human TF-1 cells to assay human IL-3.
Interleukin-4 (IL-4): IL-4 is a ~20 kDa protein with three disulfide bonds that are critical for biological activ-
ity. IL-4 is glycosylated but this is not required for biological activity. IL-4 is mainly produced by activated
CD4+ TH2 helper cells. IL-4 promotes the proliferation and differentiation of activated B-cells. In addition, IL-
4 up-regulates class II MHC antigen expression and IgE receptors. There are many cell lines available to
assay the biological activity of IL-4. Mouse MC/9 cells are used to determine the proliferative activity of
recombinant mouse IL-4 and human TF-1 cells to assay human IL-4. Rat IL-4 activity is either verified by
measuring IL-4 induced MHC antigen expression on rat splenocytes by flow cytometry or by measuring the
proliferation of the intestine endothelial cell line IEC-6.
Interleukin-5 (IL-5): IL-5 is ~32-34 kDa. The biologically active form is a disulfide-linked n-glycosylated
homodimer (glycosylation is not required for activity). IL-5 is mainly produced by T-cells and promotes the
growth and differentiation of eosinophils. Cell lines useful in IL-5 assays include B13, BCL1, T88-M, TALL-
103 and TF-1 cells. TF-1 cells are used in a proliferation assay to measure IL-5 activity.
Interleukin-6 (IL-6): IL-6 is a ~20 kDa protein with disulfide bonds that are critical for activity. IL-6 is glyco-
sylated but this is not required for biological activity. IL-6 is secreted by many cell types, but is mainly pro-
duced by stimulated monocytes, fibroblasts and epithelial cells. IL-6 is an important regulator of acute phase
reaction. It is also a B-cell differentiation factor and an activator of T-cells. There are many cell lines available
to assay the biological activity of IL-6. Mouse B9 cells are used to determine the proliferation activity of
recombinant IL-6. The cell line will react to human, mouse, swine and rat IL-6.
Interleukin-7 (IL-7): IL-7 is 17 kDa. The disulfide bonds are required for biological activity. IL-7 is secreted
from adherent bone marrow stromal cells and thymic cells. IL-7 stimulates the proliferation of pre-B and pro
B-cells. IL-7 also supports the maturation of megakaryocytes. Mouse 2E8 cells are used in a proliferation
assay to determine the activity of recombinant IL-7. The cell line will react to human and mouse IL-7.
Interleukin-8 (IL-8): IL-8 is a ~8 kDa non-glycosylated protein. IL-8 has two disulfide bonds and is a mem-
ber of the CXC chemokine family. IL-8 is produced by stimulated monocytes. It is also produced by
macrophages, fibroblasts, endothelial cells, keratinocytes, melanocytes, hepatocytes and a number of tumor
cell lines. IL-8 is chemotactic for all known types of migratory immune cells. IL-8 also inhibits the adhesion
of leukocytes to activated endothelial cells. The biological activity of IL-8 is assayed by neutrophil chemo-
taxis.

Interleukin-9 (IL-9): IL-9 is a ~16-25 kDa glycosylated protein with 5 disulfide bonds. IL-9 is secreted from
mitogen or antigen stimulated CD4 positive T helper cells. IL-9 is a cofactor in many immune processes. IL-
9 stimulates the proliferation of T helper cells. IL-9 also induces the secretion of IL-6 in mast cells. Human
IL-9 is assayed detecting the proliferation of the cell line MO7E and murine IL-9 is assayed with TS1.C3.
Interleukin-10 (IL-10): IL-10 is a ~19 kDa homodimeric protein with 2 disulfide bonds per monomer. IL-10
is secreted from activated CD8+ T-cells, B-cell lymphomas, LPS activated monocytes and mast cells. IL-10
inhibits the synthesis of a number of cytokines, such as, TNF-α, IL-1 and IL-6 from macrophages and IL-2,
IFN-γ, and TNF-β from TH1 cells. Human and mouse IL-10 is assayed by measuring the increased prolifer-
ation of the cell line MC/9 co-stimulated with a low dose of m IL-4. Rat IL-10 may either be assayed by meas-
uring the inhibition of TNF-α production from LPS stimulated rat peripheral macrophages or utilizing the
mast cell line, D36, in a proliferation assay.
Interleukin-11 (IL-11): IL-11 is a ~23 kDa non-glycosylated protein with no cysteine residues. IL-11 is main-
ly produced by bone marrow stromal cells and also mesenchymal cells. IL-11 is a cofactor in many immune
responses and in cell differentiation. For example, IL-11 works with IL-3 in stimulating the generation of
megakaryocyte colonies. In addition, IL-11 prevents cell death by apoptosis and inhibits the differentiation
of preadipocytes. Human and mouse IL-11 is assayed by measuring the proliferation of the cell lines, TF-1,
7TD1 or T1165.
Interleukin-12 (IL-12): IL-12 is a ~70 kDa heterodimeric protein comprised of a p40 and p35 subunit. The sub-
units are linked by disulfide bonds that are critical for biological activity. In addition, the p40 subunit contains
10 cysteine residues and the p35 subunit contains 7. This tertiary structure requires insect, yeast or mammalian
expression for active recombinant protein. The primary source of IL-12 is dendritic cells, peripheral lympho-
cytes, and B cells. IL-12 induces the secretion of IFN-γ, IL-2 and TNF-α and stimulates the proliferation of PHA
activated lymphocytes. Human IL-12 is assayed by measuring the proliferation of PHA activated peripheral lym-
phocytes and mouse IL-12 is by measuring the increased production of IFN-γ from mouse splenocytes.
Interleukin-13 (IL-13): IL-13 is a ~12 kDa protein with 2 disulfide bonds that are critical for biological activi-
ty. IL-13 is mainly produced by activated TH2 cells. IL-13 down regulates macrophage produced cytokines
such as TNF-α, IL-1, IL-6, IL-8 and IL-12 in response to LPS challenge or IFN-γ. IL-13 induces monocyte dif-
ferentiation and also induces B-cell differentiation and proliferation. TF-1 cells are used to assay human IL-13.
Interleukin-14 (IL-14): IL-14 is also known as high molecular weight B-cell growth factor. IL-4 is a ~50-60
kDa and is secreted from T-cells. IL-14 functions as a mitogen for activated B-cells.
Interleukin-15 (IL-15): IL-15 is a ~13 kDa protein with disulfide bonds that are critical for activity. IL-15 is gly-
cosylated but this is not required for biological activity. T-cells are a major source of IL-15. In addition, IL-15 is
secreted by astrocytes and microglia after stimulation with IL-1β, IFN-γ or TNF-α. IL-15 activity resembles
some biological activities attributed to IL-2. IL-15 induces proliferation of PMA activated peripheral blood
mononuclear cells. IL-15 also functions as a maturation factor for NK cells. Mouse CTLL cells are used to deter-
mine the proliferative activity of recombinant IL-15. The cell line will respond to human and mouse IL-15.

Interleukin-16 (IL-16): IL-16 is ~13 kDa and is known to be active as a tetramer. The major cellular source
is the epithelium, although mast cells, CD8 positive cells, CD4 cells and cosinophils are also sources. IL-16
stimulates a migratory response in CD4+ cells such as lymphocytes, monocytes and eosinophils. IL-16 activ-
ity is measured by detecting the chemotaxis of CD4+T lymphocytes.
Interleukin-17 (IL-17): IL-17 is a ~16 kDa glycosylated homodimer. CD4 positive T-cells are a major source
of IL-17. IL-17 stimulates epithelial, endothelial or fibroblastic cells to secrete IL-6, IL-8 and G-CSF. IL-17
also increases the expression of ICAM-1 in fibroblasts. IL-17 activity is measured by detecting IL-6 produc-
tion in foreskin fibroblasts.
Interleukin-18 (IL-18): IL-18 is ~18 kDa. Liver cells are a major source of IL-18. IL-18 induces IFN-γ in acti-
vated peripheral blood lymphocytes. Human IL-18 activity is measured by detecting IFN-γ secretion from
CD3 stimulated PBMCs and mouse IL-18 activity is measured by detecting IFN-γ secretion in ConA stimu-
lated murine lymph node cells.
Interleukin-19 (IL-19): IL-19 is a 18 kDa and is a recently discovered member of the IL-10 family. IL-19 is
expressed in activated kerationcytes and monocytes. IL-19 binds a receptor complex consisting of the IL-20
receptor alpha (IL-20 Rα, also known as IL-20 R1) and the IL-20 receptor beta (IL-20 Rβ or IL-20 R2). This
receptor complex is also shared by IL-20 and IL-24. IL-19 induces IL-6 and TNF-α production by monocytes,
which helps to initiate T-helper cell differentiation towards a Th2 response.
Interleukin-20 (IL-20): IL-20 is a ~17 kDa and is known to occur in skin and trachea. Activated keratinocytes and,
most-likely, monocytes express IL-20. There are two heterodimeric receptor complexes for IL-20. The first is com-
posed of IL-20 Rα and IL-20 Rβ. The second is composed of IL-22 R and IL-20β . IL-20 induces the proliferation
of multipotential hematopoietic progenitor cells, directs the differentiation and expansion of keratinocytes, and pro-
motes the release of proinflammatory mediators in keratinocytes and other cells expressing the IL-20 receptor.
Interleukin-21 (IL-21): IL-21 is a ~14 kDa and is related to IL-2, IL-4 and IL-15. IL-21 R, is a type I cytokine
receptor and is most closely related to IL-2 Rβ and IL-4 Rα. The IL-21/IL-21 R interaction appear to play
important roles in B and T cell proliferation after antigen stimulation and NK cell maturation.
Interleukin-22 (IL-22): IL-22 is a ~16 kDa. IL-22 activates STAT-1 and STAT-3 in several cell lines and upreg-
ulates the production of acute phase proteins. IL-22 is produced by normal T cells when stimulated with anti-
CD3 in humans. Mouse IL-22 is expressed in various organs upon lipopolysaccharide injection. The func-
tional IL-22 receptor complex consists of two receptor subunits, IL-22R and IL-10Rβ.
Interleukin-23 (IL-23): IL-23 is a ~62 kDa and is composed of two subunits, a p19 subunit, exclusive to IL-
23, and a p40 subunit that is shared with IL-12. p19 is expressed by activated macrophages, dendritic cells,
and T cells, but only activated macrophages and dendritic cells express p40 in parallel to produce IL-23. The
IL-23 receptor complex consists of two receptor subunits, the IL-12 receptor beta 1 subunit (IL-12 Rb1) and
the IL-23-specific receptor subunit (IL-23 R). IL-23 has biological activities that are similar to, but distinct
from IL-12. Both IL-12 and IL-23 induce proliferation and IFN-γ production by human T cells. While IL-12
acts on both naïve and memory human T cells, the effects of IL-23 is exclusive to memory T cells.

Interleukin-24 (IL-24): IL-24 is ~19 kDa and is part of the IL-10 family of cytokines. Cells known to express
IL-24 include B cells, CD4+ T cells, NK cells, lymph node dendritic cells, monocytes, melanocytes, and
melanoma cells. IL-24 appears to have multiple functions. At low concentrations, it induces type I proin-
flammatory cytokines such as IFN-γ, IL-1β, IL-12 and TNF-α on monocytes. At high concentrations, apop-
tosis occurs in tumor cells. IL-24 also has anti-angiogenic properties. It directly binds IL-24 receptors on
endothelial cells, activating STAT3 and blocking their differentiation. IL-24 binds and signals through two het-
erodimeric receptor complexes. One complex is the combination of IL-20Rα and IL-20Rβ, which is shared
with IL-19 and IL-20. The second complex is a combination of IL-22 R and IL-20 Rβ, which is shared with
IL-20.
Interleukin-26 (IL-26): IL-26 is ~19 kDa and was cloned from herpesvirus saimiri (HVS)-transformed T-cells,
but is also expressed in other virus transformed T cell lines, fresh peripheral mononuclear cells, activated NK
cells and T cells. IL-26 is a member of the IL-10 family of class II cytokines. Activation of the receptor com-
plex results in phosphorylation of STAT1 and STAT3. Although the IL-26 receptor complex is highly specific
for IL-26 and is not activated by other class II cytokines, the individual subunits of the IL-26 receptor com-
plex are components in receptor complexes for other class II cytokines. The physiological functions of IL-26
remain are under investigation.
Interleukin-27 (IL-27): IL-27 is and~50-60 kDa and belongs to the IL-6/IL-12 family of long type I cytokines.
It is composed of EBI3 (EBV-induced gene 3), a 34 kDa glycoprotein that is related to the p40 subunit of IL-
12 and IL-23, and p28, the recently cloned 28 kDa glycoprotein that is related to the p35 chain of IL-12. IL-
27 has both anti- and proinflammatory properties. As an antiinflammatory, IL-27 appears to induce negative
feedback that limits T and NK-T cell activity. IL-27 also induces an IL-12 receptor on naïve CD4+ T cells.
Interleukin-28A (IL-28A) and Interleukin-29 (IL-29): Both IL-28A and IL-29 are ~21 kDa are recently dis-
covered class II cytokine receptor ligands that are part of the IL-10 family. The expression of IL-28A, and
IL-29 is induced by virus infection or double-stranded RNA. These cytokines are functionally similar to type
I interferons, including antiviral activity and up-regulation of MHC class I antigen expression. These proteins
signal through the same heterodimeric receptor complex that is composed of the IL-10 receptor β (IL-10 Rβ)
and a novel IL-28 receptor α (IL-28 Rα, also known as IFN-γ R1). Binding to the receptor complex induces
Jak kinase activation and STAT1 and STAT2 tyrosine phosphorylation.
Interleukin-30 (IL-30): IL-30 is a 28~kDa and is a member of the long-chain 4-helix bundle cytokine family,
and EBI3, form the IL-27 heterodimer. This heterodimer is expressed by antigen-presenting cells. IL-27 trig-
gers expansion of CD4+ T-cells and helps to drive a Th1 response.
Interleukin-31 (IL-30): IL-31 is a ~16 kDa and is a part of the α-helical family of cytokines. IL-31 is prima-
rily associated with activated T-cells and expressed by Th2 rather than Th1 cells. IL-31 signals via IL-31RA
and the oncostatin M receptor. IL-31 signaling has been shown to involve the Jak/STAT pathway, the PI3
kinase/AKT cascade, and MAP kinase pathway.

Tumor necrosis factor-alpha (TNF-α): TNF-α is a ~17 kDa protein with a single disulfide bond that is not
necessary for activity. TNF is active as a homotrimer. TNF-α is secreted by macrophages, monocytes, neu-
trophils, T-cells and NK cells following LPS challenge. TNF causes cytolysis and cytostasis of many tumor
cell lines. TNF has a wide spectrum of activities, including chemotaxis of neutrophils, alteration of the
endothelium, inhibition of anticoagulatory mechanisms, and promotion of angiogenesis. TNF-α activity is
measured by detecting the cytolysis of the murine cell line L929. This cell line is appropriate for testing
human, mouse and rat TNF-α. Swine TNF-α is tested with PK-15 cells.
Tumor necrosis factor-beta (TNF-β): TNF-β is a ~17 kDa protein that forms heterodimers with LT-beta that
anchors the complex in the cell membrane. TNF-β is produced by mitogen activated T-cells and leukocytes.
Additional cell sources include fibroblasts, astrocytes, myeloma cells, endothelial cells, epithel cells and cer-
tain transformed cell lines. TNF-β induces the synthesis of GM-CSF, G-CSF and IL-1. It is a cytolytic factor
many tumor cell lines and induces reactive oxygen species in neutrophils. TNF-β activity is measured by
detecting the cytolysis of the murine cell line L929.

ELISA Kits
Background
There are four general types of ELISAs. (1) Direct ELISAs involve attachment of the antigen to the solid
phase, followed by an enzyme-labeled antibody. This type of assay generally makes measurement of crude
samples difficult, since contaminating proteins compete for plastic binding sites. (2) Indirect ELISAs also
involve attachment of the antigen to the solid phase, but in this case, the primary antibody is not labeled. An
enzyme-conjugated second antibody, directed at the first antibody, is then added. This format is used most
often for the detection of specific antibodies in sera. (3) A third type of ELISA is the Competition Assay, which
involves the simultaneous addition of ‘competing’ antibodies or proteins. The decrease in signal of samples
where the second antibody or protein is added gives a highly specific result. (4) The last type of assay is the
Sandwich ELISA. Sandwich ELISAs involve attachment of a capture antibody to a solid phase support.
Samples containing known or unknown antigen are then added in a matrix or buffer that will minimize attach-
ment to the solid phase. An enzyme-labeled antibody is then added for detection.
The Enzyme-Linked-ImmunoSorbent Assay (ELISA) method is a benchmark for quantitation of pathological

antigens and there are indeed many variations to this method. ELISAs are adaptable to high-throughput
screening because results are rapid, consistent and relatively easy to analyze. The best results have been
obtained when utilizing a Sandwich format, which combines pre-matched capture antibody, Biotin-labeled
detection antibody and our proprietary Horseradish Peroxidase conjugated Streptavidin. The resulting signal
provides data which is very sensitive and highly specific. BioSource offers a complete line of ELISAs and
ultra-sensitive ELISAs for the quantitation of human, mouse, rat, swine, and monkey cytokines, chemokines,
growth factors and adhesion molecules. For a more detailed description of ELISA theory, refer to ELISA:
Theory and Practice by J.R. Crowther, 1995, Humana Press.
Pre-Coated Sandwich ELISAs
Optimizing Pre-coated Sandwich ELISAs

There is no need for optimization of pre-coated ELISA assays from BioSource as exhaustive effort has gone
into optimizing each and every step of the assay by assay experts at BioSource. This is one of the major ben-
efits of using pre-coated plates. However, it is very important that all steps of the protocol be followed, espe-
cially the recommended washing technique and incubation times (see troubleshooting guide for more infor-
mation). For your convenience, ideal washing suggestions are listed below.
Method for Washing Pre-Coated ELISA Plates

Incomplete washing will adversely affect the test outcome. All washing must be performed with Wash Buffer
provided. Washing can be performed manually as follows: completely aspirate the liquid from all wells by
gently lowering an aspiration tip (aspiration device) into the bottom of each well. Take care not to scratch the
inside of the well.
After aspiration, fill the wells with at least 0.4 mL of diluted wash solution. Let soak for 15 to 30 seconds,
then aspirate the liquid. Repeat as directed under ASSAY METHOD. After the washing procedure, the plate is
inverted and tapped dry on absorbent tissue.

Alternatively, the wash solution may be put into a squirt bottle. If a squirt bottle is used, flood the plate with
wash buffer, completely filling all wells. After the washing procedure, the plate is inverted and tapped dry on
absorbent tissue. If using an automated washer, the operating instructions for washing equipment should be
carefully followed.
General ELISA Procedure

BioSource EASIA ELISA kits
In addition to the hundreds of ELISA kits that BioSource offers for human, mouse, rat, monkey and swine
samples, BioSource also offers specialty ELISA kits, or EASIA ELISA Kits, specifically designed for problem-
atic clinical serum samples that often produce high backgrounds using traditional ELISAs. Key features of
this product line are listed below. If you have further questions, please contact our technical service depart-
ment at 800-242-0607.
Primary Features of BioSource EASIA ELISA Kits
• 1- or 2-plate format.
• Designed for clinical use; approved for diagnostic use in Europe. Specifically designed
for use with serum, plasma and other biological fluids.
• All sample types can be read off the same standard curve.
• Kits include assayed controls, with ranges printed on the label (2 levels). Also sold sep-
arately in the catalog.
• Standards are pre-diluted, preferred by clinical researchers. No serial dilutions required.
• Utilizes F(ab')2 fragments of mAbs developed in-house to eliminate false values that
commonly appear in pathological samples, especially the rheumatoid arthritis model.
• Utilizes only mono/mono pairs. Some kits use a mAb cocktail as capture to avoid
hyper-specificity.
• Only non-neutralizing mAbs are used to eliminate interference from soluble receptor
often found in biological (clinical) samples, such as serum, plasma and synovial fluid.
• Pre-diluted standards eliminate errors made in pipetting serial dilutions or from making
the serial dilutions in an unacceptable matrix.
• Large batch sizes made in Belgian ISO-9001 approved facility.
• Tolerance on lot-to-lot variability is +5%.
• Quarterly QC testing done throughout the shelf life.
• Calibration to WHO/NIBSC.

BioSource Human EASIA ELISA Kits
Product Size Catalog # Product Size Catalog #

17-β Estradiol (E2) 96 tests KAQ0621 IL-8 96 tests KAC1301
Aggrecan/PG 96 tests KAP1461 IL-8 192 tests KAC1302
Aggrecan/PG 192 tests KAP1462 Insulin 96 tests KAQ1251
Calcitonin 96 tests KAQ0421 IP-10 96 tests KAC2361
CD14 (soluble) 96 tests KAS0231 IP-10 192 tests KAC2362
CD23 (soluble) 96 tests KAS0251 Leptin 96 tests KAC2281
CD23 (soluble) 192 tests KAS0252 Leptin 192 tests KAC2282
C-Peptide 96 tests KAQ0401 LHsp (Specific) (Luteinizing Hormone) 96 tests KAQ1311
Eotaxin/CCLII 96 tests KAC2231 LIF-HILDA 96 tests KAC1351
Eotaxin/CCLII 192 tests KAC2232 (Leukemia Inhibitory Factor/Human Interleukin for DA Cells)
FSH (Follicle Stimulating Hormone) 96 tests KAQ0841 LIF-HILDA 192 tests KAC1352
GM-CSF 96 tests KAC0901 (Leukemia Inhibitory Factor/Human Interleukin for DA Cells)
GM-CSF 192 tests KAC0902 MIG/CXCL9 96 tests KAC2431
HGF 96 tests KAC2211 MIG/CXCL9 192 tests KAC2432
HGF 192 tests KAC2212 MIP-1α/CCL3 96 tests KAC2201
hGh (Growth Hormone) 96 tests KAQ1081 MIP-1α/CCL3 192 tests KAC2202
IFN-γ 96 tests KAC1231 MIP-1β/CCL4 96 tests KAC2291
IFN-γ 192 tests KAC1232 MIP-1β/CCL4 192 tests KAC2292
IL-10 96 tests KAC1321 MMP-13 96 tests KAC2411
IL-10 192 tests KAC1322 MMP-3 96 tests KAC1541
IL-12 (p40 & p70) 96 tests KAC1561 MMP-3 192 tests KAC1542
IL-12 (p40 & p70) 192 tests KAC1562 Osteocalcin 96 tests KAQ1381
IL-12 (p70) 96 tests KAC1568 Progesterone 96 tests KAQ1451
IL-12 (p70) 192 tests KAC1569 Prolactin 96 tests KAQ1441
IL-17 96 tests KAC1591 PTH (Parathyroid Hormone) 96 tests KAQ1481
IL-17 192 tests KAC1592 RANTES/CCL5 96 tests KAC1511
IL-1α 96 tests KAC1191 RANTES/CCL5 192 tests KAC1512
IL-1α 192 tests KAC1192 Testosterone 96 tests KAQ1701
IL-1β 96 tests KAC1211 TGF-β1 (Multi-Species) 96 tests KAC1688
IL-1β 192 tests KAC1212 TGF-β1 (Multi-Species) 192 tests KAC1689
IL-1ra 96 tests KAC1181 TNF-α 192 tests KAC1752
IL-1ra 192 tests KAC1182 TNF-α 96 tests KAC1751
IL-2 96 tests KAC1241 TNF-RI (soluble) 96 tests KAC1761
IL-2 192 tests KAC1242 TNF-RI (soluble) 192 tests KAC1762
IL-4 96 tests KAC1281 TNF-RII (soluble) 96 tests KAC1771
IL-4 192 tests KAC1282 TNF-RII (soluble) 192 tests KAC1772
IL-6 192 tests KAC1262 TSH (Thyroid Stimulating Hormone) 96 tests KAQ1881
IL-6 96 tests KAC1261

Cross-reactivity of BioSource Human ELISA Kits with Non-human Primates
Human Interferon-α
Human Interferon-α CytoScreen ELISA kit (KHC4012)
Hensley, L.E., H.A. Young, P.B. Jahrling, and T.W. Geisbert. 2002. Proinflammatory response during Ebola
virus infection of primate models: possible involvement of the tumor necrosis factor receptor superfamily.
Immunol. Lett. 80:169-179.
This paper demonstrates cross-reactivity with rhesus monkey and cynomolgus monkey samples.
Human Interferon-β
Human Interferon-β EASIA ELISA kit (KAC1201)
Human IL-1β
Human IL-1β EASIA ELISA kit (KAC1211/KAC1212)
Human IL-1β CytoScreen ELISA kit (KHC0011/KHC0012)
These two kits use the same antibody format. This paper demonstrates cross-reactivity with rhesus mon-
key and cynomolgus monkey samples.
Human IL-6
Human IL-6 EASIA ELISA kit (KAC1261/KAC1262)
IL-6 CytoScreen ELISA kit (KHC0061/KHC0062)
Lozier, J. N., M.E. Metzger, R.E. Donahue, and R.A. Morgan. 1999. Adenovirus-mediated expression of
human coagulation factor IX in the rhesus macaque is associated with dose-limiting toxicity. Blood
94(12):3968-3975.
The two kits use the same antibody format. This paper demonstrates cross-reactivity with rhesus monkey
samples.
Lozier, J.N., G. Csako, T.H. Mondoro, D.M. Krizek, M.E. Metzger, R. Costello, J.G. Vostal, M.E. Rick, R.E.
Donahue, and R.A. Morgan. 2002. Toxicity of a first-generation adenoviral vector in rhesus Macaques.
Human Gene Therapy 13(1):113-124.
This paper demonstrates cross-reactivity with rhesus monkey samples.

Shean, M.K., G. Baskin, D. Sullivan, J. Schurr, D.E. Cavender, J.E. Shellito, P.O. Schwarzenberger, and J.K.
Kolls. 2000. Immunomodulation and adenoviral gene transfer to the lungs of nonhuman primates. Human
Gene Therapy 11:1047-1055.
Immul. Lett. 80:169-179.
Human IL-8
Human IL-8 EASIA ELISA kit (KAC1301/KAC1302)
Human IL-8 CytoScreeen ELISA kit (KHC0081/KHC0082)
Shean, M.K., G. Baskin, D. Sullivan, J. Schurr, D.E. Cavender, J.E. Shellito, P.O. Schwarzenberger, and J.K.
Kolls. 2000. Immunomodulation and adenoviral gene transfer to the lungs of nonhuman primates. Human
Gene Therapy 11:1047-1055.
The two kits use the same antibody format.
Human RANTES
Human RANTES CytoScreen ELISA kit (KHC1031/KHC1032)

CytoSet™ Antibody Pairs
Background
CytoSet Antibody Pairs from BioSource are lot-matched, pretitered antibody pairs designed for measuring
human, mouse, rat and swine cytokine levels in cell culture supernatants. The detection antibody is conju-
gated to biotin. Each CytoSet Antibody Pairs kit includes:
• Monoclonal or oligoclonal capture antibody, sufficient for 10 or 40 plates

• Biotin-conjugated monoclonal detecting antibody, sufficient for 10 or 40 plates
• Single-use recombinant standards, sufficient for 10 or 40 plates
• Streptavidin-HRP conjugate
• General ELISA protocol and lot-specific procedure
• Lot-specific example standard curve
The sensitivities of CytoSet Antibody Pairs will be generally comparable to the corresponding EASIA or
Cytoscreen™ parameters and can be estimated from the standard curve included in the package insert.
Typical Standard Curve

Rat MIP-2 CytoSet (Cat. #CRC1033)
Rat MIP-2 CytoSet

4-
3-
2-
1-
0-
10 100 1000
Rat MIP-2 (pg/mL)
The standard curve shown above was generated using the CytoSet Buffer Set (Cat. #CNB0011) from
BioSource. As a general rule, BioSource’s antibody pair reagents are optimized such that the standard curve
results in a high O.D. of approximately 3.0 to 4.0.

Recommended Buffers and Solutions for Use with CytoSet Antibody Pairs
The BioSource CytoSet Buffer Set (Cat. #CNB0011) containing Coating Buffers A and B, Assay Buffer,
Substrate Solution (TMB), Stop Solution, and Wash Buffer is recommended for use with CytoSet Antibody
Pairs.
1. Coating Buffer A: Coating Buffer A (Cat. # CB07100) from BioSource is recommended.*

8.0 g NaCl, 1.13 g Na2HPO4, 0.2 g Na2HPO4, 0.2 g KCl; q.s. to 1.0 L with
distilled H2O, pH to 7.4.
2. Coating Buffer B: Coating Buffer B (Cat. # CB01100) from BioSource is recommended.*
4.3 g NaHCO3, 5.3 g Na2CO3; q.s. to 1.0 L with distilled H2O, pH to 9.4.
3. Assay Buffer: Assay Buffer (Cat. # DS98200) from BioSource is recommended.*
8.0 g NaCl, 1.13 g Na2HPO4, 0.2 g KH2PO4, 0.2 g KCl, 5.0 g bovine serum
albumin (fraction V), 1 mL Tween 20; q.s. to 1.0 L with distilled H2O,
pH to 7.4.
4. Wash Buffer: Wash Buffer (Cat. # WB01) from BioSource is recommended.*
9.0 g NaCl, 1 mL Tween 20; q.s. to 1.0 L with distilled H2O, pH to 7.4.
5. Substrate Solution: TMB (Cat. # SB01) from BioSource is recommended.*
Tetramethylbenzidine (TMB) and Hydrogen Peroxide.
6. Stop Solution: Stop Solution (Cat.# SS01100) from BioSource is recommended.*
1.8 N H2SO4.
* Alternate choice listed below.
Please note that all components of the CytoSet Buffer Set (Cat. #CNB0011) may be purchased as individual
components using the catalog numbers listed above next to each component.
Recommended Assay Procedure for CytoSet Antibody Pairs

BioSource has recently revised its recommended assay procedure for antibody Pairs. We recommend that
you use the following procedure when running CytoSet Antibody pairs.
1. Prepare coating solution by diluting the coating antibody. See “coating antibody” section of technical
data sheet for the recommended coating antibody dilution.
2. Coat plates with 100 µL per well of the coating solution. Cover plates and incubate overnight (12-18 hr.)
at 2-8ºC.
3. Aspirate wells and wash 1X with >400 µL of Wash Buffer (Cat. #WB01) per well. Following last wash,
invert and tap on absorbent paper to remove excess liquid.
4. Block plate with 300 µL per well of Assay Buffer (Cat. #CNB0011) for 1 hour at room temperature.
5. Aspirate, invert, and tap on absorbent paper to remove excess liquid.
6. Prepare standards and sample dilutions in Assay Buffer (or in a diluent that most closely matches the
matrix of your sample). For recommended dilutions and storage of the standard, see “standard” section
of the technical data sheet.
7. Pipette 100 µL of standards, samples and controls (in duplicate) into designated wells.
8. Immediately following step 7, add 50 µL of the working detection antibody into each well. For recom-

mended dilutions, see “detection antibody” section of the technical data sheet. Gently tap the plate on
the side 10 times to mix. Cover plate and incubate for 2 hours at room temperature.
9. Aspirate and wash 5X using the method in step 3.
10. Add 100 µL of the working streptavidin-HRP solution into each well. For recommended dilutions, see
“streptavidin-HRP conjugate” section of the technical data sheet. Cover plate and incubate for 30 min-
utes at room temperature.
11. Aspirate and wash 5X using the method in step 3.
12. Add 100 µL of the TMB (Cat. #SB01) substrate to each well. Incubate plate without a plate cover for 30
minutes in the dark at room temperature.
13. Add 100 µL of Stop Solution (Cat. #SS01100) to each well.
14. Measure absorbance at 450 nm (reference absorbance: 650 nm) within 30 minutes of adding Stop
Solution. Calculate results using a log-log or 4-parameter curve fit.
Plate Coating Tips

1. Dilute coating antibody in Coating Buffer (Cat. #CBO7100 or CBO1100) to the concentration recom-
mended on the accompanying CytoSet Antibody Pairs technical information sheet. Refer to vial label for
concentration of coating antibody. Avoid polystyrene containers when storing or diluting antibody. If the
standard curve signals are not as high as desired, higher concentrations of coating antibody may be
tried.
2. Add 100 µL of diluted coating antibody per well to polystyrene microplates. (e.g., Dynex Immulon 2 HB
or Nunc Maxisorp). Other polystyrene plates may also be suitable.
3. Cover the plates and incubate overnight (12 to 18 hours) at 2-8°C. Incubating for longer periods of time
such as over the weekend should not cause any problems.
4. Aspirate the coating antibody from the wells and tap on absorbent paper to remove excess liquid.
5. Add >400 µL per well of Wash Buffer, incubate for 15-30 seconds, and aspirate.
6. Add 300 µL of Assay Buffer (Cat. #DS98200) to each well. Cover the plates and incubate for 1 hour at
room temperature. Excessive blocking should be avoided. If backgrounds are higher than desired, try
substituting either casein or calf serum in place of the BSA found in the Blocking Solution.
7. Aspirate the Assay Buffer for blocking from the wells and tap on absorbent paper to remove excess liq-
uid. Plates can now be used. Alternatively, if the plates are not to be used immediately, allow to dry
overnight at room temperature and store at 2-8°C in a plastic bag with desiccant for up to a week.
Optimization of CytoSet Antibody Pairs for ELISA

Preparation
1. Read the entire technical information sheet. This sheet describes the kit components in detail and the
specific procedure to be followed in order to obtain the example standard curve illustrated.
2. Visually inspect all materials and check expiration date.
3. Determine the number of plates to be run. It is recommended to coat only the number of plates needed
for each sample run.
4. Collect all materials required for the assay. The quality of the microtiter plates will influence the per-
formance of the assay. Good results have been obtained with the following microplates: Nunc Maxisorp
(Catalog # 468667) and Dynex Immulon 2 HB (Catalog #6506).
5. Prepare fresh buffers. For CytoSet, use of NaN3 in the streptavidin dilution buffer must be avoided.

6. Check pipette calibration and if an automated washing device is used, check that it is working properly.
A squirt bottle will suffice for washing if automated equipment is not available.
7. Check the microplate reader for the appropriate filter. The substrate recommended requires capacity to
read at a wavelength of 450 nm with a reference filter of 650 nm.
Antibodies
Certainly, one of the most important characteristics of a good assay is the selection of the antibodies and a
suitable antigen. Once they have been selected, the appropriate titers and volumes must be chosen. A stan-
dard curve range of 0-2 ng/mL is generally acceptable for measurement of cytokines in most sample types.
Both the capture and detecting antibodies must be independently titered and working volumes chosen.
Recommendations are included in all lot-specific CytoSet technical information sheets.
1. The capture antibody can be tested in concentrations ranging from 0.5-10 µg/mL. Serial dilutions should
be prepared starting with 10 µg/mL and the coating volume should be 100-200 µL.
2. The biotinylated detection antibody should be tested in concentrations ranging from 0.05-2.0 µg/mL. The
volume of detection antibody can be 50 to 100 µL.
Divide the plate in 4 parts according to 4 different coating concentrations to be tested. Each coating con-
centration will be tested together with 3 different detecting antibody concentrations and 4 standard points
(ranging from zero = standard 0 and increasing to standard 3 = highest standard value). The SAV-HRP con-
centration is maintained at a fixed concentration during this experiment.
The best working conditions will satisfy the following criteria:
-standard 0: < 0.2 O.D.

-standard 3: 1.5-2.5 O.D.
-standard 1
: ≥2
standard 0
Incubations: Time and Temperature

Incubation times and temperatures for all steps should be empirically determined. In general, the temperature
of incubation will contribute to the determination of the incubation time. For example, if the standard/sample
incubation is performed at 2-8°C, then usually an overnight incubation will be required to obtain a desirable
signal. When incubations are performed at 37°C, then much shorter incubation times are necessary.
1. Coating of the microwells may be performed at room temperature or at 4°C. Optimal saturation of the
plastic should occur over 4-48 hours. Note, however, that various plastics from different vendors must
also be tested for optimal performance.
2. The temperature of the detection antibody incubation can also be varied from 4-37°C. The incubation
time is dependent on sample recovery and desired signal for both a separate and combined assay.
Generally, 30 to 120 minutes is enough time for the detection antibody incubation step.

Signal Intensity
With the protocols given, the non-specific signal (zero value) should be less than 0.2 O.D. units. To lower the
non-specific signal:
1. Decrease the incubation times or volumes of either the sample and/or the biotinylated detection antibody.
2. Improve the washing procedure. Use the wash buffer recommended in the package insert. When washing,
fill the wells by delivering a forceful stream of buffer into the wells. Allow the wells to soak for 15-30 sec-
onds. Aspirate the wash buffer completely. Repeat 2-5 times. Following the final aspiration, tap the plate
forcefully on absorbent paper until all of the residual buffer is removed. Proceed immediately to the next
step. Omitting Tween-20 from the wash buffer has also been observed to reduce the non-specific signal.
3. Increase the incubation time and concentration of the blocking solution. The use of casein as a blocking
agent may reduce non-specific signal.
4. Optimize the Standard Diluent/Assay Buffer by decreasing or increasing the amount of animal serum or
BSA. (Refer to Buffers section.)
Buffers
For best results, BioSource recommends that the BioSource CytoSet Buffer Set (Cat. #CNB0011) is used with
CytoSet Antibody Pairs.
1. The coating buffer most commonly used is carbonate buffer (Coating Buffer B). Phosphate buffers have
also worked well in BioSource’s labs. The ionic strength (200-400 nM) and pH (3-10 pH units) of these
buffers can be altered to achieve more efficient capture antibody binding.
2. Assay diluent buffers usually contain a serum protein which helps to mimic the proteins contained in
sample types. In the CytoSet Antibody Pairs assay systems, bovine serum albumin is the protein rec-
ommended. In some cases where recovery is poor (e.g., less than 80%), increasing the concentration of
BSA or addition of animal serum will increase recovery.
3. Streptavidin diluents affect not only the stability of the enzyme, but also non-specific binding and the rate
of the color change. Horseradish Peroxidase, the enzyme used in CytoSet Antibody Pairs, is active over
a range of pH, but if the pH is too low, the HRP may become unstable. Detergent, temperature and molar-
ity of the buffers are all variables which, when altered, can affect the performance of HRP.
Sensitivity
Sensitivity, or Minimum Detectable Concentration (MDC), is confirmed by assaying a minimum of 20 repli-
cates of the zero standard in a single assay. The concentration interpolated from the standard curve of the
average O.D.s for the standard 0 replicates + 2 SD is the MDC. This value represents the lowest value read
from the standard curve that can be statistically differentiated from zero.
Assay sensitivity may be estimated from the example standard curve provided on the technical information
sheet. If the specific procedure is closely followed, the example standard curve can be duplicated. The
desired level of sensitivity and range of the curve may affect the concentrations of reagents used. For exam-
ple, if the range of the curve is too narrow, a higher concentration of streptavidin may be required. The opti-
cal limitations of the plate reader must also be considered.
Assay precision and background affect the assay sensitivity. Precision can be improved with the use of cal-
ibrated pipettes, careful technique and vigorous washing. Choose assay conditions which show the highest
signal to noise ratio.

Standards
Additional vials of standard may be purchased using the part number listed on the technical data sheet.
The standards provided in the Human CytoSet Antibody Pairs are calibrated against NIBSC/WHO standard refer-
ence preparations (if available). For best accuracy, the use of these standards is recommended for calibrating the
assay. The use of proteins other than those included with the CytoSet Antibody Pairs to calibrate the assay may
produce different recovery values. Expressing results in NIBSC/WHO units will minimize confusion when com-
paring inter-laboratory data. To avoid any discrepancy, compare weight expressed standard preparations for
other sources of protein by converting from mass to activity (in Units or International Units) as follows:
Human
1 ng GM-CSF CytoSet standard = 7.3 IU NIBSC 88/646
0.3 pg/mL HGF CytoSet standard = 1 mIU/mL NIBSC 95/556
1 µg IFN-γ CytoSet standard = 20,000 IU NIBSC 87/586
1 pg IL-1α CytoSet standard = 0.2 mU NIBSC 86/632
1 µg IL-1β CytoSet standard = 100,000 IU NIBSC 86/680
1 pg IL-2 CytoSet standard = 0.02 U NIBSC 86/504
1 pg IL-2R CytoSet standard = 37mIU NIBSC 97/600
1 µg IL-3 CytoSet standard = 1700IU NIBSC 91/510
1 µg IL-4 CytoSet standard = 10,000 U NIBSC 88/656
1 pg IL-6 CytoSet standard = 100 mIU NIBSC 89/548
1 ng IL-8 CytoSet standard = 7.1 IU NIBSC 89/520
1 ng IL-10 CytoSet standard = 7 IU NIBSC 92/516
1 µg IL-13 CytoSet standard = 1000 U NIBSC 94/622
1 µg IP-10 CytoSet standard = 14,600 U NIBSC 89/514
1 pg MIP-1α CytoSet standard = 0.25mU NIBSC 92/518
1 µg TGF-β1 CytoSet standard = 14,600 U NIBSC 89/514
1 µg TNF-α CytoSet standard = 40,000 IU NIBSC 87/650
Mouse
43 pg IFN-γ CytoSet standard = 1U NIH Gg02-901-533
1 ng IL-1β CytoSet standard = 450 U NIBSC 93/668
1 ng IL-2 CytoSet standard = 30 U NIBSC 93/566
1 µg IL-3 CytoSet standard = 140,000U NIBSC 91/662
1 ng IL-4 CytoSet standard = 3.8 U NIBSC 91/656
1 µg TGF-β1 CytoSet standard = 14,600U NIBSC 89/514
1 ng TNF-α CytoSet standard = 180 U NIBSC 88/532

Frequently Asked Questions About CytoSet Antibody Pairs:
Q. How long are the antibodies stable after dilution?

A. All diluted antibody reagents should be used as soon as possible after diluting.
Q. How long are the assay buffers stable after preparation?

A. Pre-made buffers and solutions are available for purchase from BioSource (Cat. #CNB0011 – CytoSet
Buffer Set) and are guaranteed for 6 months. However, It is recommended that investigators prepare
fresh dilutions, if making their own alternate buffers.
Q. What is the limit of sensitivity that can be attained by CytoSet Antibody Pairs?
A. The sensitivity limit can be easily estimated from the standard curve presented on the technical data
sheet. Lot performance will vary slightly. However, buffer formulations employed by the investigator can
potentially alter the slope and the standard curve which ultimately affects sensitivity. This is specifically
why BioSource recommends using the CytoSet Buffer Set (Cat. #CNB0011) when using antibody pairs
from BioSource. These reagents are pre-optimized for use with CytoSet antibody pairs.
Q. Are components available separately?

A Yes, components may be ordered by the individual catalog number listed on the technical data sheet.
Technical information sheets may be easily viewed on the web at www.biosource.com. Or, contact tech-
nical customer support at 800-242-0607.
Q. Can the CytoSet Buffer Set be used for all species of CytoSet Antibody Pairs?
A. Yes, the universal buffers and solutions (Cat. #CNB0011) are pre-optimized and can be used with any
CytoSet antibody pair for best results.

Methods for ELISA Sample Preparation
General Guidelines for the Production of Serum and Plasma for Cytokine Analysis
Serum is the liquid fraction of whole blood that is collected after the blood is allowed to clot. The clot is
removed by centrifugation and the resulting supernatant, designated serum, is carefully removed using a
Pasteur pipette. Plasma is produced when whole blood is collected in tubes that are treated with an anti-
coagulant. The blood does not clot in the plasma tube. The cells are removed by centrifugation. The super-
natant, designated plasma, is carefully removed from the cell pellet using a Pasteur pipette.
Procedures for the Production of Serum and Plasma

Serum preparation
Collect whole blood in a covered test tube. If commercially available tubes are to be used, the researcher
should use the red topped tubes. These are available from Becton Dickinson. BD's trade name for the blood
handling tubes is Vacutainer. After collection of the whole blood, allow the blood to clot by leaving it undis-
turbed at room temperature. This usually takes 15-30 minutes. Remove the clot by centrifuging at 1,000-
2,000 x g for 10 minutes in a refrigerated centrifuge. The resulting supernatant is designated serum.
Following centrifugation, it is important to immediately transfer the liquid component (=serum) into a clean
polypropylene tube using a Pasteur pipette. The samples should be maintained at 2-8˚C while handling. If the
serum is not analyzed immediately, the serum should be apportioned into 0.5 mL aliquots and stored and
transported at –20˚C or lower. It is important to avoid freeze/thaw cycles because this is detrimental to many
serum components. Samples which are hemolyzed, icteric, or lipemic can invalidate certain tests.
Plasma preparation
Collect whole blood into commercially available anti-coagulant treated tube, such as EDTA treated (lavender
tops) or citrate treated (light blue tops). Heparinized tubes (green tops) are indicted for some applications;
however, heparin can often be contaminated with endotoxin and endotoxin can stimulate white blood cells to
release cytokines. Cells are removed from plasma by centrifugation for 10 minutes at 1,000-2,000 x g using
a refrigerated centrifuge. Centrifugation for 15 minutes at 2,000 x g depletes platelets in the plasma sample.
The resulting supernatant is designated plasma. Following centrifugation, it is important to immediately
transfer the liquid component (=plasma) into a clean polypropylene tube using a Pasteur pipette. The sam-
ples should be maintained at 2-8˚C while handling. If the plasma is not analyzed immediately, the plasma
should be apportioned into 0.5 mL aliquots and stored and transported at –20˚C, or lower. It is important to
avoid freeze/thaw cycles. Samples which are hemolyzed, icteric, or lipemic can invalidate certain tests.

There are other commercially available tubes for blood sample collection. BioSource has not evaluated some
of these tubes for compatibility with our ELISA kits. The commercially available serum tubes are as follows:
Red: No anticoagulant.
Red with black: Treated with gel to help to separate the clot (not evaluated).
The commercially available plasma tubes are as follows:

Lavender: Treated with EDTA.
Blue: Treated with citrate.
Green: Treated with heparin.
Grey: Treated with potassium oxalate/sodium fluoride (not evaluated).
Yellow: Treated with ACD (not evaluated).
References:
1. Henry, J.B. (1979) Clinical Diagnosis and Management by Laboratory Methods, Volume 1, W.B Saunders Company,
Philadelphia, PA, p. 60.
2. Thavasu, P.W., S. Longhurst, S.P. Joel, M.L. Slevin, and F.R. Balkwill (1992) Measuring cytokine levels in blood.
Importance of anticoagulants, processing, and storage conditions. J. Immunol. Methods 153:115-124.
Preparing Tissue Culture Samples

1. Following the end of the desired cell culture time, pipette medium into microcentrifuge
tube and immediately put on ice.
2. Centrifuge at 1,400 RPM for 1 minute in microcentrifuge.
3. Remove supernatant fluid and aliquot into a clean microcentrifuge tube.
4. Store at -80ºC until ready for use in ELISA.
Sample Preparation Using BioSource’s Tissue Extraction Buffer (catalog #FNN0071)

BioSource offers a tissue extraction buffer for total protein extractions from various tissue sample types.
Primary tissues are valuable tools for the study of intracellular and extracellular markers which characterize
disease states. We have developed a protocol for rapid isolation of cytokines and signaling molecules from
intact tissue. This method is for total protein extraction and makes use of a non-abrasive tissue extraction
reagent. Tissue samples as little as 10 mg may be extracted using this protocol. This method is sensitive and
allows for the detection of disease-associated fluctuations of biomarkers. This is an effective system for the
extraction of proteins from a variety of tissue types. Heart, lung, kidney, spleen, brain, liver, thymus, and
smooth muscle tissues have all been successfully extracted with this protocol. The prepared extracts are com-
patible with a variety of immunoassays, such as ELISA and Luminex, as well as with all conventional protein
assays. Our extraction method is inexpensive, versatile, and can be completed in less than fifteen minutes.

Preparing the Tissue Extraction Buffer for Use
The tissue extraction buffer must be supplemented with protease inhibitor cocktail (not included) just prior
to use. We recommend Sigma catalog #P-2714. Reconstitute and add according to manufacturer’s instruc-
tions. The stability of the protease inhibitor-supplemented cell extraction buffer is 24 hours at 2-8ºC.
General Protocol
This protocol has been successfully applied to several tissue types. However, some optimization may be
required.
1. Add protease inhibitors to the BioSource Tissue Extraction Buffer as needed just prior to use.
2. Weigh tissue sample.
3. Add 10 mL of the Tissue Extraction Buffer per 1 g of tissue.
4. Homogenize.
5. Centrifuge the sample at 10,000 RPM for 5 minutes to pellet the tissue debris.
6. Collect supernatant fluid, aliquot, and freeze at -80ºC. Avoid multiple freeze/thaws.

Method for Isolating of Intracellular and
Extracellular Proteins from Whole Tissue
In this study, we have used the BioSource mouse VEGF ELISA to evaluate various mouse tissue homogenates
using BioSource’s Tissue Extraction Buffer (Cat. #FNN0071) and have demonstrated that this assay provides
a highly sensitive method for the detection of endogenous levels of VEGF in all types of mouse tissue.
Summary
Vascular endothelial growth factor (VEGF) plays a central role in tumorigenesis. Subjects with invasive can-
cer manifest significant elevations in both the endogenous tissue and the serum levels of VEGF; therefore,
VEGF serves as an important metastatic biomarker. BioSource has developed an Enzyme-Linked
Immunosorbent Assay (ELISA) to evaluate mouse VEGF levels in serum, plasma, and tissue homogenates.
This study aims at correlating the circulating and the endogenous tissue levels of this marker in tumor mod-
els. The BioSource mouse VEGF ELISA utilizes highly-specific antibodies as capture and detector, and
exhibits no cross-reactivity with other cytokines such as Eotaxin, G-CSF, GM-CSF, IFN-γ, IL-1β, IL-2, IL-4,
IL-5, IL-6, IL-10, IL-12, and TNF-α. The detection range of the assay is 7.8-500 pg/mL, and the system meas-
ures both recombinant and natural mouse VEGF proteins with sensitivity of less than 5 pg/mL. This
immunoassay has intra-assay and inter-assay coefficients of variation of 4.4% and 7.2%, respectively.
BioSource has demonstrated that this ELISA measures the titrated levels of recombinant VEGF protein in
exact parallel to those of natural VEGF protein. In this assay, the recovery of mouse VEGF from different bio-
logical matrices is greater than 95%. BioSource’s mouse VEGF ELISA provides a fast, reliable method for the
quantitative determination of mouse VEGF in a variety of natural systems, including serum, plasma, cell cul-
ture supernatants, and tissue extracts.
VEGF Background
VEGF was first discovered two decades ago as the endothelial tumor permeability factor. This cytokine has
since been found to play a central role in normal as well as disease-associated angiogenesis and vasculariza-
tion. VEGF is present in measurable quantities in most organs. Macrophages and smooth muscle cells com-
prise the main sources of VEGF in normal healthy individuals, although lower levels of this cytokine are also
present in healthy lung, heart, and liver tissues. A direct correlation between VEGF expression and angiogen-
esis has long been established. VEGF levels are increased in all physiological states which require vascular
permeability. Moreover, continuous infusion of VEGF in normal brain tissue has been shown to induce angio-
genesis. The pathophysiological conditions which upregulate VEGF expression include hypoxia, cancer, burn,
tissue injury, and ionizing radiation. This cytokine is also a critical mediator of developmentally-regulated
angiogenesis and lymphangiogenesis as manifested by its pronounced levels during pregnancy and early
post-natal stages. The most notable elevations of circulating VEGF have been documented in patients with
invasive cancer. These findings are exploited in the treatment of solid tumors. In clinical studies, local injec-
tions of anti-VEGF-specific antibodies into solid tumors have effectively diminished the rate of growth of the
malignant tissue. BioSource has developed a highly sensitive Enzyme Linked Immunosorbent Assay (ELISA)
that allows for the quantitative determination of natural mouse VEGF in serum, plasma, cell culture super-
natants, whole cell lysates, and tissue homogenates. This assay employs a sandwich ELISA method and was
developed using highly-purified antibodies as capture and detector. The BioSource mouse VEGF ELISA takes
less time than commercially available immunoassays.

Reagents
Antibodies
Polyclonal antibodies to VEGF, G-CSF, and EGF were produced by immunizing rabbits with baculovirus- or E.
coli-expressed recombinant proteins and purifying sera with protein A gel chromatography. Rabbit polyclonal
antibodies to G-CSF and EGF were produced by immunization with the E. coli-expressed recombinant pro-
teins. All polyclonal antibodies were purified using the protein A gel purification method. Monoclonal anti-
bodies to mouse KC were raised by immunizing hamsters with E. coli-expressed recombinant KC protein, fol-
lowed by purification with protein G gel. Human IL-13 monoclonal antibody was produced by immunizing
mice with the E. coli-expressed recombinant protein and purifying the antibody with protein gel.
Preparation of Tissue Extracts (using BioSource’s Tissue Extraction Buffer, Cat. #FNN0071)
Mouse tissues obtained from normal healthy mice were snap-frozen in liquid nitrogen immediately following
dissection and maintained at -80°C. To prepare tissue extracts, frozen tissues were weighed and placed in 50
mL conical tubes. Subsequently, the BioSource tissue extraction buffer (with protease inhibitors) at ratio of
10 mL per 1g of tissue was added to each tube. Tissues were solubilized using a Janke & Kunkel GmbH &
Co KG (Staufen, Germany) IKA Labortechnik Ultra-Turrex T250 homogenizer. The homogenates were then
transferred to round-bottom tubes and centrifuged at 10,000 RPM for 5 minutes Supernatants were
removed, aliqoutted, and frozen at -80°C.
Neutralization Assay
Specificity of the immunoassay towards tissue mouse VEGF was demonstrated by pre-incubating normal-
ized volumes of the tissue extract with 10 µg of each anti-mouse VEGF polyclonal, anti-mouse G-CSF poly-
clonal, anti-mouse EGF polyclonal, anti-mouse KC monoclonal, and anti-human IL-13 monoconal antibodies.
All antibody-treated samples, as well as untreated controls, were then incubated at 37°C for 30 minutes and
analyzed on the mouse VEGF immunoassay.
Mouse VEGF ELISA
To prepare the BioSource mouse VEGF ELISAs (Cat. #KMG0111 and #KMG0112), purified polyclonal anti-
body against mouse VEGF was coated onto microtiter plates and blocked using blocking buffer. The detec-
tion antibody for the immunoassay was prepared by biotinylation of yet another highly purified anti-mouse
VEGF polyclonal antibody. The immunoassay was performed by loading 100 µL tissue extracts or the stan-
dards into the wells, followed by addition of 50 µL of the detection antibody. Samples were incubated at room
temperature for 2 hours and then washed 4 times. Next, 100 µL of strepavidin-HRP conjugate was added to
each well, and plates were incubated for 30 minutes at room temperature. Plates were once again washed 4
times, and 100 µL of stabilized chromogen was added to each well. After 30 minutes of incubation at room
temperature, the reaction was terminated by the addition of 100 µL of the Stop Solution to each well. The
absorbance values were measured at the wavelength of 450 nm.

Measurement of VEGF Levels in Normal Mouse Tissue
Tissue Type VEGF / g Tissue VEGF / mg Total Protein

Brain 0.6 ng 0.01 ng
Heart 1.8 ng 0.03 ng
Kidney 10.4 ng 0.10 ng
Liver 7.5 ng 0.06 ng
Lung 24.1 ng 0.61 ng
Smooth Muscle 8.8 ng 0.24 ng
Spleen 0.6 ng 0.01 ng
Thymus 0.8 ng 0.03 ng
Levels of VEGF in various mouse tissues obtained from apparently healthy mice were evaluated on the
BioSource mouse VEGF ELISA. VEGF quantities in each tissue were normalized against both the starting tis-
sue mass and the total protein concentration of each tissue. Variations in the measured levels of VEGF in dif-
ferent tissues correspond to those reported for each tissue, indicating that this assay is a suitable method
for quantitative analysis of tissue homogenates.

Protocol Summary

Specificity of BioSource Mouse VEGF ELISA
4.5 Untreated
4 Rbt x Ms VEGF PAB

3.5
3 Rat x Ms KC MAB
2.5 Rbt x Ms G-CSF

2 PAB
Rat x Hu IL-13 MAB
1.5
1 Rbt x Ms EGF PAB
0.5
Rbt x Hu HGF PAB
0
0 20 40 60 80 100
VEGF (pg)
Normalized volumes of the extracts from the mouse smooth muscle were pre-incubated with 10 µg of mouse
VEGF, G-CSF, and EGF polyclonal or mouse KC, and human IL-13 monoclonal antibodies for 30 min at 37°C.
Untreated control samples were also included in this study. Samples were then evaluated at various dilutions
on the mouse VEGF ELISA. As shown in this figure, the signal was completely blocked by rabbit anti-mouse
VEGF antibody, but not by any of the unrelated antibodies. This demonstrates that this assay is specific for
the tissue mouse VEGF protein.

Linearity of Dilution of Mouse Tissue Extracts on Mouse VEGF ELISA
Smooth Muscle R2 = 0.9947
600
500
400
300
200
100
0
0 100 200 300 400 500 600
Expected Value (pg/mL)
Liver R2 = 0.9986
200
150
100
50
0
0 50 100 150 200
Kidney R 2 = 0.9952
600
500
400
300
200
100
0
0 100 200 300 400 500 600
Heart R 2 = 0.9855
80
60
40
20
0
0 10 20 30 40 50 60 70
Samples of various mouse tissue extracts were two-fold serially diluted in the ELISA standard diluent buffer
and evaluated on the mouse VEGF ELISA. The correlation coefficient was determined by plotting measured
values against their respective expected values. The linearity of dilution of all tissue mouse VEGF produces
a correlation coefficient of greater than 0.9.

Parallelism between Recombinant and Natural Mouse VEGF
4 Standard
3.5 3T3 Cell

Supernatant
3
SVEC4-10 Cell
2.5 Supernatant
Lung
2
1.5 Smooth Muscle
1 Liver
0.5
Kidney
0
0 100 200 300 400 500 600 Heart
Mouse VEGF Concentration (pg/mL)
Aliquots of various mouse tissue extracts as well as culture supernatants from 3T3 and SVEC-4-10 cells (as
controls) were two-fold serially diluted in the standard diluent buffer of the kit and analyzed on the mouse
VEGF ELISA. Parallelism between recombinant VEGF in the standard and natural VEGF in various tissue and
cell culture supernatants was demonstrated by plotting absorbance values at 450 nm against measured con-
centrations of mouse VEGF for each dilution.
Conclusion
1. The BioSource Mouse VEGF ELISA offers a quantitative method for the measurement of natural VEGF in
mouse tissue.
2. This immunoassay is specific for the tissue mouse VEGF protein.
3. The linearity of dilution of mouse VEGF extracted from all tissues produces a correlation coefficient of
>0.9.
4. This assay measures recombinant mouse VEGF protein in parallel with the natural protein.
5. Different buffer formulations can influence the efficacy of VEGF recovery from tissue extracts.

Cell Extraction Buffer Protocol (for use in ELISA)
BioSource also offers cell extraction buffer (Cat. #FNN0011) for use in ELISA.
This cell extraction buffer must be supplemented with 1 mM PMSF (not included) and protease inhibitor
cocktail (not included) just prior to use.
• For the PMSF addition, we recommend making a 0.3 M stock in DMSO, and adding sufficient volume
for a final concentration of 1 mM (i.e., 17 µL per 5 mL cell extraction buffer). PMSF is very unstable and
must be added just prior to use, even if added previously.
• For the protease inhibitor cocktail addition, we recommend Sigma Cat. # P-2714, reconstituted accord-
ing to the manufacturer’s instructions, and adding 250 µL per 5 mL cell extraction buffer. The stability of
protease inhibitor-supplemented cell extraction buffer is 24 hours at 4°C.
Protocol: This protocol has been successfully applied to several cell lines and various tissue types. Some
optimization may be required for specific applications.
1. Collect cells in PBS by centrifugation (non-adherent) or scraping from culture flasks (adherent).
2. Wash cells twice with cold PBS.
3. Remove and discard the supernatant and collect the cell pellet.
4. Lyse the cell pellet in cell extraction buffer for 30 minutes, on ice, with vortexing at 10 minute intervals.
The volume of cell extraction buffer depends on the cell number and expression of target protein and
level of phosphorylation. A suitable starting concentration is 108 cells per mL extraction buffer.
5. Transfer the extract to microcentrifuge tubes and centrifuge at 13,000 rpm for 10 minutes at 4oC.
6. Aliquot the clear lysate to clean microfuge tubes. These samples are ready for assay. Lysates can be
stored at -80°C. Avoid multiple freeze/thaws.

Comparison of BioSource Tissue Extraction Buffers
Comparison of BIOSOURCE Tissue Extraction Buffers

in Mouse Smooth Muscle Tissue
350
300
250
Buffer 1
200 Buffer 2
150 Buffer 3
Buffer 4
100
50
0
1:2 1:4 1:8 1:16 1:32 1:64 1:128
Dilutions of Tissue Extracts
Comparison of BIOSOURCE Tissue Extraction Buffers

in Mouse Liver Tissue
380
330
280
230 Buffer 1
Buffer 2
180
Buffer 3
130
Buffer 4
80
30
-20
1:16 1:32 1:64 1:128
Dilutions of Tissue Extracts
Buffer 1: Competitor’s buffer

Buffer 3: BioSource’s Cell Extraction Buffer (Cat. #FNN0011)
To compare various tissue extraction buffers offered by BioSource, equal quantities of tissue from a homog-
enous mass of mouse smooth muscle or liver were extracted in equal volumes of four different cell extrac-
tion buffers. These tissue/cell extracts were analyzed at multiple dilutions in the Mouse VEGF ELISA. (Top)
Comparison of various dilutions of the extracts from smooth muscle tissue; (Bottom) Comparison of vari-
ous dilutions of the extracts from liver tissue.

Preparing Rat Liver Samples for Cytokine Analysis
1. Remove liver aseptically.
2. Flash freeze liver in foil packets using liquid nitrogen. Store at -80ºC.
3. Crush liver sample on dry ice and store in a pre-chilled 5 mL culture tube.
4. Add about 100 µg tissue (enough to almost cover round portion of a 5 mL culture tube)
to a new chilled tube.
5. Add 1 mL PBS with 10 µL protease inhibitors (Sigma cat. #P8340). Chill this solution using wet ice.
6. Homogenize at low speed for ~20 seconds. Make sure to keep cool and keep samples on wet ice.
7. Transfer samples into 1.7 mL microcentrifuge tubes.
8. Centrifuge at 14000 x g at 4ºC for 15 minutes.
9. Remove and aliquot supernatant. We recommend to make several 50 µL aliquots.
10. Before running ELISA, dilute protein 1:100 and run a Bradford total protein assay.
11. Dilute the samples to equal concentrations and then run ELISA as described in BioSource protocols.
Cardiac Puncture for Blood Sample in Rats – Open Chest Method for Cytokine Analysis
1. Rat should be fully anesthetized (e.g., unresponsive to toe pinch).
2. Open chest method
a. Have ready a syringe (3-10 mL) containing ~0.3 mL air and with a 20 ga needle attached.
b. Make an incision at the level of the xiphoid process, and hold the xiphoid with a hemostat or forceps.
c. Beginning at the xiphoid, cut along the full length of the sternum just to the rat’s right of the midline,
then make a lateral incision through the chest wall laterally to the rat’s left, just cranial to the
diaphragm. Do this quickly, as there will be arterial bleeding from the chest wall incisions.
d. Continuing to hold the xiphoid, retract the chest wall flap laterally, and expose the heart with blunt dis-
section of the pleura and pericardium. If necessary, make a lateral incision to the right in the chest
wall (there should be adequate exposure of the heart without the second lateral incision if the initial
midline incision is slightly to the right of the sternum).
e. Insert the needle into the right ventricle and apply gentle negative pressure to the syringe to fill the
syringe with blood. Excessive negative pressure will collapse the chamber. The right atrium also works
well for drawing blood, but the left side of the heart is more difficult.
Cardiac Puncture for Blood Sample in Rats – Closed Chest Method for Cytokine Analysis
2. Closed chest method
a. Have ready a syringe (3-10 mL) containing ~0.3 mL air and with a 20 ga needle attached.
b. Make an incision in the skin and abdominal wall, caudal to the sternum.
c. Using a hemostat or thumb forceps applied to the xiphoid process, retract the sternum cranially until
the apex of the heart is visible against the diaphragm.
d. Insert the needle through the diaphragm and into the heart, applying gentle negative pressure once
the heart has been punctured, and reposition the needle as necessary until blood flows into the
syringe. Avoid excessive negative pressure, as it will collapse the heart chamber onto the needle tip.

Method for Perfusion to Flush Nonadherent Cells From the Pulmonary Circulation in Rats.
2. Euthanize the rat by exsanguination. The easiest way to do this is by cutting the abdominal aorta. Avoid
cardiac puncture, as the puncture site will leak during the perfusion procedure.
3. Open the chest widely, taking care not to puncture the lungs. Cut through the entire sternum at the mid-
line; lifting the sternum with forceps and keeping the scissor tips horizontal will lessen the chance of
inadvertent lung puncture. Next make lateral incisions in the chest wall in both directions from the mid-
line incision. Retract one side of the chest wall laterally, bluntly dissect the pleura to free up the lung,
gently push the lung away from the chest wall with a Q-tip, and cut the chest wall vertically along the
spinal processes. Repeat for the other half of the chest wall. Be careful not to puncture the lung with the
sharp edges of the cut ribs.
4. Place vascular clamps or tie ligatures on both the superior and inferior vena cavae to prevent backflow
of perfusion buffer into the systemic venous system.
5. It helps to use magnification (dissecting microscope or eye magnifiers) for the remaining steps.
6. With a scalpel or small scissors, make an incision in the LEFT ventricle of the heart, making certain that
the heart chamber is open to the outside.
7. Prepare a 10 mL syringe filled with PBS, with a stopcock and a small gauge needle (25 or 27 ga)
attached. Have a second PBS-filled syringe available in case it is needed. Insert the needle into the RIGHT
ventricle, and gently push the PBS into the pulmonary circulation. You should see the right heart and pul-
monary artery swell a little bit, and fluid should immediately begin to flow from the left ventricle incision.
Continue to push PBS through until the lungs are white and the fluid coming out of the left ventricle is
completely clear- this usually takes 10-15 mL of buffer. The small needle will prevent you from pushing
in the buffer too rapidly, and also will have fewer problems with leaks than with larger needles.
8. Another option that works well for the perfusion, but that takes more time, is to cannulate the pulmonary
artery. This can be done by introducing a plastic catheter through the right heart and into the pulmonary
artery, and ligating the pulmonary artery to hold the catheter in place. With this method it is not neces-
sary to clamp the vena cavae. Buffer may be pushed into the catheter with a syringe, taking care not to
create excessive pressure in the pulmonary system, or with a gravity feed of buffer not more than about
10 cm above heart level.

ELISA Troubleshooting Guide
Problem: Elevated Background

Cause: Insufficient washing and/or draining of wells after washing. Solution containing either biotin or
SAV-HRP can elevate the background if residual is left in the well.
Solution: Wash according to the protocol. At the end of each washing step, invert plate on absorbent tissue
on countertop and allow to completely drain and tap forcefully if necessary to remove residual.
Cause: Contamination of substrate solution with metal ions or oxidizing reagents.

Solution: Use distilled/deionized water for dilution of wash buffer and use plastic equipment. DO NOT
COVER plate with foil.
Cause: Contamination of pipette, dispensing reservoir or substrate solution with SAV-HRP conjugate.
Solution: Do not use chromogen that appears blue prior to dispensing onto the plate. Obtain new vial of
chromogen.
Cause: Improper dilution of SAV-HRP working dilution. SAV-HRP concentrate contains 50% glycerol,
is viscous and therefore, difficult to accurately measure.
Solution: Warm solution of SAV-HRP concentrate to room temperature, draw up slowly and wipe tip with
tissue to remove excess.
Cause: Chromogen exposed to light prior to use, resulting in a blue color.

Solution: Keep chromogen in vial until ready to dispense into plate and then pour into a reservoir to pre-
vent contamination of the vial with equipment. DO NOT COVER plate with foil.
Cause: Incorrect incubation times and/or temperatures.

Solution: Follow protocol (room temperature is 25±2°C).
Cause: Evaporation of wells during incubations.

Solution: Securely seal plate with plate cover during all incubations.
Cause: Improperly set up blanks.

Solution: Follow protocol when designating blank wells. Blank wells contain only chromogen and stop
solution and results should be subtracted from all other wells.
Cause: Incorrect amounts of SAV-HRP or chromogen dispensed into wells.

Solution: Follow protocol for volumes to dispense and check pipette calibration.
Cause: Incorrect dilution of standard stock solution; intermediary dilutions not followed correctly.
Solution: Follow the protocol instructions regarding the dilution of the standard.
Cause: Standards diluted in serum, culture supernatant or other.

Solution: Dilute standards ONLY in standard diluent buffer provided in the kit.

Problem: Elevated Sample/Standard ODs
Cause: Incorrect dilution of the SAV-HRP conjugate.
kim-wipe to remove excess. Dilute ONLY in SAV diluent provided.
Cause: Plate covered with foil during chromogen step.

Solution: Do not cover plate with foil. Place plate in a drawer or some other light-free chamber during
last incubation step.
Cause: Incubation times extended.

Solution: Follow incubation times outlined in protocol.
Cause: Incubations carried out at 37°C when RT is dictated.

Solution: Perform incubations at RT=25±2°C when instructed in the protocol.
Cause: Contamination of pipette, dispensing reservoir or substrate solution with SAV-HRP conjugate.
Solution: Do not use chromogen that appears blue prior to dispensing onto the plate. Obtain new vial of
chromogen.
Cause: Chromogen exposed to light prior to use.

Solution: Exposure of the chromogen to light causes the solution to change from clear to blue. Keep
chromogen in vial until ready to dispense into plate and then pour into a reservoir to prevent
contamination of the vial with equipment. DO NOT COVER the plate with foil.
Problem: Poor Standard Curve

Cause: Improper preparation of standard stock solution.
Solution: Dilute lyophilized standard as directed by the vial label only with the standard diluent buffer.
Cause: Standard was not diluted with the standard diluent buffer.
Solution: Standard MUST be diluted with the standard diluent buffer.
Cause: Inadequate washing and draining of wells.

Solution: Wash according to the protocol. At the end of each washing step, invert plate on absorbent tissue
on countertop and allow to completely drain and tap forcefully if necessary to remove residual.
Cause: Errors in pipetting the standard or subsequent steps.

Solution: Always dispense into wells quickly and in the same order. Do not touch the pipette tip on the indi-
vidual microwells when dispensing. Use calibrated pipettes and the appropriate tips for that device.
Cause: Reagents (lyophilized standard, standard diluent buffer, etc.) from different kits, either differ-
ent cytokine or different lot number, were substituted.
Solution: NEVER substitute any components from another kit.

ELISA Troubleshooting Guide (continued)
Problem: Weak/No Color Develops

Cause: Reagents not at RT (25±2°C) at start of assay.
Solution: Allow ALL reagents to warm to RT prior to commencing assay.
Cause: Incorrect storage of components, e.g. not stored at 2-8°C.

Solution: Store all components exactly as directed in protocol and on labels.
Cause: Incubation of SAV-HRP step at 37°C when RT is directed.

Solution: Follow instructions for SAV-HRP step incubation temperature given in the protocol.
Cause: Omission of any incubation steps.

Solution: Follow incubation steps outlined in the protocol.
Cause: Standards diluted in serum, culture medium, or other solution.

Solution: Standards must be diluted in the standard diluent buffer provided in the kit.
Cause: Microwells were allowed to dry out.

Solution: Do not allow microwells to sit uncovered. After loading, place adhesive strip over plate to pro-
tect from evaporation. Unused microwells should be returned to the pouch with desiccant and
sealed and stored at 2-8°C.
Cause: Incorrect chromogen/stop solution used.

Solution: Use only the chromogen and stop solution contained in the kit.
Cause: Excessively cool laboratory temperature, i.e., <22°C.

Solution: Adjust laboratory temperature to 25±2°C.
Cause: Reagents have expired.

Solution: Check expiration dates upon receipt of kit and use kit prior to expiration.
Cause: Wells have been scratched with pipette tip or washing tips.
Solution: Use caution when dispensing and aspirating into and out of microwells.
Cause: Plate read at incorrect wavelength.

Solution: The correct wavelength for CytoScreen and EASIA kits is 450 nm.
Cause: Incorrect dilution of the SAV-HRP conjugate.

tissue to remove excess. Dilute ONLY in SAV diluent provided.

Problem: Weak/No Color Develops (continued)
Cause: Inadequate collection system for analyte being measured.
Solution: Some analytes require special handling to make accurate measurements, e.g., collection of
blood in heparinized vacutainer tubes will hinder accurate measurement of TNF-α.
Cause: Attempt to measure analyte in a matrix for which the CytoScreen assay has not been optimized
Solution: Please contact BioSource Technical Service for advice when using non-validated sample types.
Cause: Standard Diluent Buffer added to ALL wells when it should only be added to wells designated
as the "0" standard.
Solution: Follow the protocol and only add standard diluent to the "0" wells and to the samples where it
is required or to samples producing signals greater than that of the highest standard.
Cause: Sodium azide-containing Standard Diluent Buffer used to dilute the SAV-HRP concentrate.
Solution: Dilute the SAV-HRP concentrate with the SAV-HRP Diluent.
Cause: Incorrect dilution of standard stock solution; intermediary dilutions not followed correctly.
Solution: Follow the protocol instructions regarding the dilution of the standard.
Cause: Working SAV-HRP solution made up longer than 15 minutes before use in assay.
Solution: Use the diluted SAV-HRP within 15 minutes of Dilute the SAV-HRP concentration up to 15
minutes prior to using in the assay.
Solution: Use the diluted SAV-HRP within 15 minutes of Dilute the SAV-HRP concentration up to 15
minutes prior to using in the assay.
Problem: Poor Precision

Cause: Errors in pipetting the standards, samples or subsequent steps.
Solution: Always dispense into wells quickly and in the same order. Do not touch the pipette tip on the indi-
vidual microwells when dispensing. Use calibrated pipettes and the appropriate tips for that device.
Cause: Wells have been scratched with pipette tip or washing tips.
Solution: Use caution when dispensing and aspirating into and out of microwells.
Cause: Liquid has been transferred from well to well during incubations.
Solution: Do not use an orbital shaker during incubations. Peel adhesive plate cover off carefully.
Cause: Incorrect volumes of materials dispensed into microwells.

Solution: Follow protocol for reagent dispensing volumes. Check calibration of pipettes.

ELISA Troubleshooting Guide (continued)
Problem: Poor Precision (continued)

Cause: Standard was diluted with the serum, culture medium or other buffer.
Solution: Standard MUST be diluted with the standard diluent buffer provided in the kit.
Cause: Particulates or precipitates found in the samples.

Solution: Remove any particulates/ precipitates by centrifugation prior to dispensing into the assay.
Cause: Dirty microwells-visible debris within or on bottom of microwells.

Solution: Inspect microwells and invert plate to remove debris. Wipe bottom of plate with a absorbent
tissue after each wash step. Never insert tissue into microwells.
Cause: "Edge effect"-due to uneven temperature from the outer edge wells to the wells in the center of
the plate.
Solution: Seal the plate completely with cover during incubations and place plate in center of incubator
when 37°C incubation is indicated.

CytoSet Antibody Pairs Troubleshooting Guide
Problem: Poor Standard Curve

Cause: Improper preparation of standard stock solution.
Solution: Dilute lyophilized standard as directed with the suggested diluent buffer, or in a diluent that
most closely matches the matrix of your sample.
Cause: Improper dilutions from standard stock solution.

Solution: Check your calculation for the dilution.
Cause: Reagents used beyond expiration date.

Solution: Check the expiry date listed in the information sheet.
Cause: Use of non-calibrated external recombinant protein preparation as standard.

Solution: Calibrate against NIBSC/WHO reference preparation standard.
Problem: High Background

Cause: Wrong saturation component.
Solution: Use a saturation buffer with a higher protein content (such as BSA or casein).
Cause: Incubation time is too long.

Solution: Reduce incubation time.
Cause: Incubation temperature is too high.

Solution: Reduce incubation temperature.
Cause: Evaporation of fluid during incubation (37°C).

Solution: Cover the plates during incubation.
Cause: Concentration of biotinylated detection antibody and/or SAV-HRP is too high.

Solution: Block titrate biotin and SAV-HRP conjugates.
Cause: Inadequate washing after the streptavidin-HRP step.

Solution: Verify function of automated plate washer.
Cause: Contamination of substrate with metal ions or oxidizing agents.

Solution: Always use distilled water.
Cause: Substrate exposed to light.

Solution: Check for unusual appearance in all components.
Cause: Degraded streptavidin-HRP.

Solution: Check for unusual appearance in all components.

CytoSet Antibody Pairs Troubleshooting Guide (continued)
Problem: Weak/no color development

Solution: Use the diluted SAV-HRP within 15 minutes of Dilute the SAV-HRP concentration up to 15 min-
utes prior to using in the assay.
Cause: Used reagents beyond expiration date.

Solution: Use reagents which are not expired.
Cause: Plate allowed to dry out between incubations.

Solution: Keep plate covered during all incubations; perform pipetting steps in a timely manner to avoid
excess exposure.
Cause: Incorrect storage of components.

Solution: See product insert for storage recommendations.
Cause: Reagents not at RT (18-25°C) at start of assay.

Solution: Allow all reagents to warm to RT prior starting the assay.
Cause: Wrong coating concentration(too low).

Solution: Titrate coating antibody concentration.
Cause: Incorrect volume of reagents dispensed.

Solution: Review assay steps to avoid error.
Cause: Omission of any incubation step.

Solution: Review assay steps to avoid error.
Cause: Contamination of one of the reagents.

Solution: Check for unusual appearance.
Cause: SAV-HRP incubation step at 37°C when RT is directed

Solution: Follow instructions for SAV-HRP step incubation temperature.
Cause: Incorrect chromogen/stop solution used.

Solution: Use chromogen/stop solutions which are recommended in the protocol.
Cause: Used buffer containing azide which is not compatible with HRP.
Solution: The use of azide in the assay should be avoided.
Cause: Plate read at incorrect wave length.

Solution: Read plate at correct wave length of 450 nm..

Problem: Poor precision
Cause: Incorrect volume of reagents dispensed.
Solution: Follow protocol for reagent dispensing volumes.
Cause: Errors in pipetting the standards, samples or subsequent steps.

Solution: Check pipette for calibration and leaking.
Cause: Bottom of microwells scratched with pipette tip or washing tips.

Solution: Repeat assay using new wells; avoid scratching the microwells
Cause: Particulates or precipitates found in the samples prior to dispensing into the assay.
Solution: Remove any particulates/precipitates by centrifugation.
Cause: Improper washing.

Solution: Verify proper function of washing device.
Cause: Transfer of liquid from one well to the other by shaking too vigorously when shaking required.
Solution: Check for correct rotator RPM.
Cause: Unequal evaporation of fluids.

Solution: Cover the plate during incubation.
Cause: Repetitive use of tips for several samples or different reagents.

Solution: Use fresh tips for each sample or reagent transfer.

ELISA Kit Selected References
Human IFN-γ
Monitoring of Anti-Vaccine CD4 T-cell Frequencies in Melanoma Patients Vaccinated with a MAGE-3 Protein.
Zhang, Y., et al., J. Immunol., Feb 2005; 174:2404-2411.
CD4+ T-cell Responses to SSX-4 in Melanoma Patients.

Ayyoub, M., et al., J. Immunol., Apr 2005; 174:5092-5099.
Human IL-2
Molecular Basis for the Potency of IL-10-Deficient Dendritic Cells as a Highly Efficient APC System for Activating TH1
Response.
He, Q., et al., J. Immunol., Apr 2005; 174:4860-4869.
Human IL-7
Dendritic Cells Induce MUC1 Expression and Polarization on Human T Cells by an IL-7-Dependent Mechanism.
Vasir, B., et al., J. Immunol., Feb 2005; 174:2376-2386.
Human IL-10
Myocardial Ischaemia and the Inflammatory Response: Release of Heat Shock Protein 70 After Myocardial Infarction.
Dybdahl, B., et al., Heart, Mar 2005; 91:299-304.
Human SAA
Identification of Serum Amyloid A as a Biomarker to Distinguish Prostate Cancer Patients with Bone Lesions.
Le, L., et al., Clin. Chem., Apr 2005; 51:695-707.
Human TNF-α
Plasmacytoid Dendritic Cells Control TLR7 Sensitivity of Naive B Cells via Type I IFN.
Bekeredjian-Ding, I.B., et al., J. Immunol., Apr 2005; 174:4043-4050.
Mouse KC/IL-8
Febrile-Range Hyperthermia Augments Neutrophil Accumulation and Enhances Lung Injury in Experimental Gram-Negative
Bacterial Pneumonia.
Rice, P., et al., J. Immunol., Mar 2005; 174:3676-3685.
Mouse IL-9
Unconjugated Bilirubin Inhibits VCAM-1-Mediated Transendothelial Leukocyte Migration.
Keshavan, P., et al., J. Immunol., Mar 2005; 174:3709-3718.
Rat IL-1β
Postischemic Gene Transfer of IL-10 Protects Against Both Focal and Global Brain Ischemia.
Ooboshi, H., et al., Circulation, Feb 2005; 111:913-919.
Rat IL-2
NK Cells Inhibit T-cell Proliferation via p21-Mediated Cell Cycle Arrest.
Trivedi, P.P., et al., J. Immunol., Apr 2005; 174:4590-4597.

Rat IL-12 (p40 and p70)
TLR Activation Synergizes with Kilham Rat Virus Infection to Induce Diabetes in BBDR Rats.
Zipris, E., et al., J. Immunol., Jan 2005; 174:131-142.
Rat IL-18
Inhibition of IL-18 reduces myeloperoxidase activity and prevents edema in intestine following alcohol and burn injury.
Rana, S.N., et al., J. Leukoc. Biol., May 2005; 77:719-728.
Rat TNF-α
Effect of Early Phase Adjuvant Arthritis on Hepatic P450 Enzymes and Pharmacokinetics of Verapamil: An Alternative
Approach to the Use of an Animal Model of Inflammation ffor Pharmacokinetic Studies.
Ling, S. and Jamali, F., Drug Metab. Dispos., Apr 2005; 33:579-586.
Monkey IL-12 (p40 and p70)

Immunomodulatory Effects of HSV-2 Infection on Immature Macaque Dendritic Cells Modify Innate and Adaptive Responses.
Peretti, S., et al., Blood, Apr 2005; 10.1182/blood-2004-12-4899.
CytoSet Antibody Pairs Selected References
Human IFN-γ
Chlamydia trachomatis-Specific Human CD8+ T Cells Show Two Patterns of Antigen Recognition.
Matyszak, M., et al., Infect. Immun., Aug 2004; 72:4357-4367.
Human IL-1β
Insulin-Like Growth Factor-I Stimulates IL-10 Production in Human T Cells.
Kooijman, R., et al., J. Leukoc. Biol., Oct 2004; 76:862-867.
Human IL-6
Inhibitors of TLR-9 Act on Multiple Cell Subsets in Mouse and Man In Vitro and Prevent Death In Vivo from Systemic
Inflammation.
Duramad, O., et al., J. Immunol., May 2005; 174:5193-5200.
Human IL-8
C-Reactive Protein-Induced In Vitro Endothelial Cell Activation Is an Artefact Caused by Azide and Lipopolysaccharide.
Karolina, T., et al., Thromb. Vasc. Biol., Jun 2005; 25:1225-1230.
Human IL-10
Inflammatory Markers and Physical Performance in Older Persons: The InCHIANTI Study.
Matteo, C., et al., J. Gerontol. A Biol. Sci. Med. Sci., Mar 2004; 59:242-248.
Human IL-12 (p40 and p70)

Inhibitors of TLR-9 Act on Multiple Cell Subsets in Mouse and Man In Vitro and Prevent Death In Vivo from Systemic
Inflammation.
Omar, D., et al., J. Immunol., May 2005; 174: 5193-5200.

Human IL-15
Bile Acid Aspiration and the Development of Bronchiolitis Obliterans After Lung Transplantation.
D’Ovidio, F., et al., J. Thorac. Cardiovasc. Surg., May 2005; 129:1144 1152.
Human TNF-α
An Analysis of Oxygen Consumption and Oxygen Delivery in Euthermic Infants After Cardiopulmonary Bypass With Modified
Ultrafiltration.
Li, J., et al., Ann. Thorac. Surg., Oct 2004; 78:1389-1396.
Mouse IL-1β and Mouse IL-6

Decorin Reverses the Repressive Effect of Autocrine-Produced TGF-β on Mouse Macrophage Activation.
Comalada, M., et al., J. Immunol., May 2003; 170:4450-4456.
Rat IL-10
Methylenedioxymethamphetamine Suppresses Production of the Proinflammatory Cytokine Tumor Necrosis Factor-α
Independent of a -Adrenoceptor-Mediated Increase in IL-10.
Connor, T., et al., J. Pharmacol. Exp. Ther., Jan 2005; 312:134-143.
Swine IL-4
Immune Responses of Pigs After Experimental Infection with a European Strain of Porcine Reproductive and Respiratory
Syndrome Virus.
Diaz, I., et al., J. Gen. Virol., Jul 2005; 86:1943-1951.
Managing Editor: Dr. Valerie Bressler-Hill

Technical Editor: Lance Mikus
Acknowledgements:
BioSource would like to thank Dr. Louis Rosenthal and Dr. Ronald Sorkness of the University of Wisconsin,
Madison, for their contribution in developing rat blood samples and rat lung samples for cytokine analysis.
Thank you to Risë Kwake for her technical review.

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Table of Contents

SEE PRODUCT REFERENCES, PROTOCOLS AT WWW.BIOSOURCE.COM | TECH.SUPPORT@BIOSOURCE.COM 1

2 TECHNICAL | CUSTOMER SERVICES 800.242.0607

Cytokine actions may be grouped into five broad areas:

• Development of cellular and humoral immune responses

• Interleukins – Regulate interactions between leukocytes

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Interleukin Primary Activity Principal Source

4 TECHNICAL | CUSTOMER SERVICES 800.242.0607

Interleukin Primary Activity Principal Source

Interferons Primary Activity Principal Source

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Tumor Primary Activity Principal Source

6 TECHNICAL | CUSTOMER SERVICES 800.242.0607

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8 TECHNICAL | CUSTOMER SERVICES 800.242.0607

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A. Proinflammatory Cytokines (TNF, IL-1, and IL-6)

A. Proinflammatory Cytokines (TNF, IL-1, and IL-6)

B. Chemokines (IL-8 and many others)

D. Immunomodulatory Cytokines (IL-2, IL-4, IL-5, IFN-γ and others)

10 TECHNICAL | CUSTOMER SERVICES 800.242.0607

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12 TECHNICAL | CUSTOMER SERVICES 800.242.0607

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14 TECHNICAL | CUSTOMER SERVICES 800.242.0607

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16 TECHNICAL | CUSTOMER SERVICES 800.242.0607

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The Enzyme-Linked-ImmunoSorbent Assay (ELISA) method is a benchmark for quantitation of pathological

Pre-Coated Sandwich ELISAs

Optimizing Pre-coated Sandwich ELISAs

Method for Washing Pre-Coated ELISA Plates

18 TECHNICAL | CUSTOMER SERVICES 800.242.0607

General ELISA Procedure

SEE PRODUCT REFERENCES, PROTOCOLS AT WWW.BIOSOURCE.COM | TECH.SUPPORT@BIOSOURCE.COM 19

Primary Features of BioSource EASIA ELISA Kits

• Standards are pre-diluted, preferred by clinical researchers. No serial dilutions required.

• Large batch sizes made in Belgian ISO-9001 approved facility.

• Tolerance on lot-to-lot variability is +5%.

• Quarterly QC testing done throughout the shelf life.

20 TECHNICAL | CUSTOMER SERVICES 800.242.0607

Product Size Catalog # Product Size Catalog #

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This paper demonstrates cross-reactivity with rhesus monkey samples.

22 TECHNICAL | CUSTOMER SERVICES 800.242.0607

This paper demonstrates cross-reactivity with rhesus monkey samples.

The two kits use the same antibody format.

This paper demonstrates cross-reactivity with rhesus monkey samples.

SEE PRODUCT REFERENCES, PROTOCOLS AT WWW.BIOSOURCE.COM | TECH.SUPPORT@BIOSOURCE.COM 23

• Monoclonal or oligoclonal capture antibody, sufficient for 10 or 40 plates

Typical Standard Curve

Rat MIP-2 CytoSet

24 TECHNICAL | CUSTOMER SERVICES 800.242.0607

1. Coating Buffer A: Coating Buffer A (Cat. # CB07100) from BioSource is recommended.*

Recommended Assay Procedure for CytoSet Antibody Pairs

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Plate Coating Tips

Optimization of CytoSet Antibody Pairs for ELISA

26 TECHNICAL | CUSTOMER SERVICES 800.242.0607

The best working conditions will satisfy the following criteria:

-standard 0: < 0.2 O.D.

Incubations: Time and Temperature