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I.                  INTRODUCTION 

Recently, protein microarray making use of new developments in protein engineering and detection physics have begun to emerge. The major focus of this technology is to understand drug discovery, molecular diagnostic applications and signal pathways. The basic construction of such protein chips has some similarities to cDNA microarrays, such as a glass or plastic surface dotted with an array of molecules. These molecules can be DNA or antibodies that are designed to capture proteins. With fluorescent markers or other methods of detection revealing the spots that have captured these proteins, protein microarrays are being used as powerful tools in high-throughput proteomics and drug screening. It lead to useful information for all cell function, including cell proliferation, cell death and differentiation, as well as maintenance of health status and development of disease controlled by many genes and signaling pathways. While technological innovations such as cDNA microarrays have enabled the analysis of genetic material in miniaturized test formats, the more delicate nature of protein structures has hindered the development of such devices for the analysis of proteins. However, almost all cell functions are executed by proteins, which can’t be studied by RNA or DNA alone. Experimental analysis clearly shows a disparity between the relative expression levels of mRNA and their corresponding proteins. Therefore, it’s critical to analyze the protein profile. Currently, two-dimensional electrophoresis coupled with mass spectrometry is the mainstream approach to analyzing multiple protein expression levels. However, the requirement of sophisticated devices and the lack of quantitative measurements method of analysis of multiple protein expression levels has been available to researchers until now.

Our Cytokine Ab NewArray is the commercially available cytokine protein array system. Scientists can rapidly and accurately identify the expression profiles of multiple cytokines in several hours inexpensively.

The Cytokine Ab NewArray kit provides a simple array format, and highly sensitive approach to simultaneously detect multiple cytokine expression levels from conditioned media, patient’s sera and other sources. This Technology’s approach has several advantages over cytokine detection by traditional ELISA.

First and most important one is that our approach can simultaneously detect many cytokines simple and efficiently. Secondly, the sensitivity of most cytokine is higher to be detected at pg/ml levels. As low as 10 pg/ml of IL-2 can be detected in NewArray format. Furthermore, the detection range is much greater than ELISA. For example, the detection range of IL-2 varies from 10 to 100,000 pg/ml, whereas the detection range varies only within 100-1,000 folds in a typical ELISA. Therefore, the detection range is about 100-fold greater in protein array in a typical ELISA. The variation is lower than ELSIA. As detection by densitometry, the variation between two spots ranged from 0 to 10% in duplicated experiments. In contrast, variation about 20% in ELISA is much higher. Finally, the system is much quicker and can be much quicker and can be much easier to adapt to high-throughput format.

Pathway-specific array systems allow investigators to focus on the specific problem and are becoming an increasingly powerful tool in cDNA microarray system. Cytokine Ab NewArray is particularly useful compared with the cytokine cDNA microarray system.

Besides the ability to detect protein expression, this system is more accurate reflection of active levels because it only detects secreted cytokines, and no amplification is needed. Furthermore, it’s much simpler, faster, environmentally friendlier, and more sensitive.

Simultaneous detection of multiple cytokines undoubtedly provides a powerful tool to study cytokines. Cytokines play an important role in innate immunity, apoptosis, angiogenesis, cell growth, and differentiation. Cytokines are involved in most disease processes, including cancer and cardiac diseases. The interaction between cytokines and the cellular immune system is a dynamic process. The interactions of positive and negative stimuli, and positive as well as negative regulatory loops are complex and often involve multiple cytokines.

How does Cytokine Ab NewArray work?

n           Immobilize cytokine antibody to slide.

n           Add samples（serum, supernate of cell culture, etc.）

n           Add biotinylated detecting antibody.

n           Add Cy3 or Cy5-conjugated streptavidin.

 

Descriptions of the applications of each cytokine

IFN-γ	● Antiviral, anti-proliferative, immuno-modulatory. ● Induce cell cycle arrest and apoptosis. ● Depression is associated with IFN-r production. ● Induce accumulation of the amyloid β precursor protein.
IL-1b	● Stimulation of T cells and antigen-presenting cells. ● B-cell growth and antibody production. ● Promotes hematopoiesis (blood cell formation). ● Induce inflammatory responses. ● Augments corticosteroid release, induce fever and shivering.
IL-2	● Also known as T-cell growth factor (TGF). ● Promotes proliferation and differentiation of additional CD4 cells, B-cells, and activates macrophages and oligodendrocytes. ● Proliferation of activated T cells.
IL-6	● Stimulate the production of acute phase proteins in the liver. ● Particularly play an important role in inducing B-cells to differentiate into antibody forming cells. ● Synergistic effects with IL-1 or TNF-α. ● Regulate fibroblast collagen and extracellular matrix protein generation.
*IL-8	● Enhance inflammation. ● A powerful inducer of chemotaxis for neutrophil cell.
IL-10	● Down-regulates antiviral responses by inhibiting the production of IFN-r, Antigen presentation, and macrophage production of IL-1, IL-6, & TNF-α. ● It is very important in B-cell activation. ● Inhibits inflammatory and immune responses ● Promote IgE mediated allergic responses
▲IL-12p70	● Play a pivotal role in cell mediated immunity ● Sustain long-term CTL responses ● Enhance Th1 development primarily by its ability to induce IFN-gamma production by NK and Th1 cells
*MCP-1	● Monocyte chemoattractant protein. ● Inflammatory response. ● In concert with other cytokines in cutaneous inflammation. ● Can recruit dendritic and Langerhans cells to the skin. ● Few cells produce MCP-1 constitutively, many cell types express MCP-1. ● Upon cytokine stimulation and in inflammatory diseases, including psoriasis, rheumatoid arthritis, and atherosclerosis.
RANTES	● Chemo-attractant for peripheral blood monocytes ● Selectively attract T cells of the CD4+/CD45RO+phenotype in vitro ● Wound repair was characterized by a large and sustained induction of RANTES. ● Essentially attract memory T cells to the site of inflammation as well as for the release of histamine and prostaglandin D2 from basophil.
*TGF-β	● Stimulates significant collagen and elastin production. ● Activation of TGF-b may be involved with driving scar tissue formation.
TNF-α	● Control cell death. ● Inflammatory related.

*: for Human Cytokine Ab NewArray Kit, ▲: for Mouse, Rat Cytokine Ab NewArray Kit only.

 II.               PRODUCT DESCRIPTION

 Cytokine Ab NewArray  uses specially modified glass slide（with 12 well format）as the platform for the cytokine antibody array. The surface characteristics provide excellent performance for Cytokine Ab NewArray  as a powerful tool for cytokine detection.

It is a novel protein array designed to screen biological samples for the presence of 10 different cytokines simultaneously. The antibodies spotted in each array have affinities for the following cytokines:

*: for Human Cytokine Ab NewArray Kit, ▲: for Mouse, Rat Cytokine Ab NewArray Kit only.

Cytokine detection is accomplished by the addition of a streptavidin-linked fluorophore. The positive control landing lights, spotted within each array, allow for verification that the assay worked.    ( See Table 2 on page  for a map of the array)

Each of the four slides provided with Cytokine Ab NewArray  has 12 identical arrays for 10 monoclonal capture antibodies spotted in six-repeated. They could be used to compare the presence of antigen in treatment and control sample s or among different treatment samples. Quantitative analysis of the sample(s) is possible by using purified antigen(s) on some arrays within a slide to generate a standard curve.

  

III.           KIT COMPONENTS

 l           Cytokine Ab NewArray  Slide (containing 4 chips each)

l           0.6ml 1 X Biotinylated Anti-Cytokine Antibody Cocktail (for 4 assays)

l           10X Blocking Buffer (20ml)

l           10X Wash Buffer I ( 20ml )

l           10X Wash Buffer II (20ml)

l           4 Incubation Chambers (Twelve-well chambers)

l           Analysis Software CD (Optional)

l           4 Foil Bags & Stick Paper(Optional)

Note: Cytokine Ab NewArray Wash / Block Buffer is supplied as a 10X concentrate. Dilute each 1ml volume with 9 ml of ddH₂0 to achieve a working 1X solution.

 IV.            GENERAL CONSIDERATIONS

 A.     Cross-reactivity

Due to the nature of immunochemical binding reactions, non-specific cross-reactivity of factors in biological samples, media or in the Biotinylated detection antibody cocktail may occur with the arrayed capture antibodies. The Blocking Solution included in the Cytokine Ab NewArray  kit is designed to reduce this cross-reactivity.

 B.     Standard Curves

Several arrays on each slice can be reserved to run a standard curve to quantify level of cytokine antigen. A standard curve must be run for each antigen to be quantified. Purified antigens can be combined to generate multiple standard curves, simultaneously, within the same set of arrays.

To generate a standard curve, make serial dilutions of the purified cytokine antigen(s) in the same media or diluents as the sample. Refer to Table 1 below, for the suggested standard curve range for individual cytokines.

 Table 1: Standard Curve Range for Individual Cytokines

 

Cytokine	Limit of Quantification1	Suggested Range for Standard Curve2
IFN-g	< 100 pg/ml	30-3,000 pg/ml
IL-1b	<  10 pg/ml	3-1,000 pg/ml
IL-2	<  30 pg/ml	10-3,000 pg/ml
IL-6	<  10 pg/ml	3-3,000 pg/ml
*IL-8	<  10 pg/ml	3-1,000 pg/ml
IL-10	< 300 pg/ml	100-10,000 pg/ml
▲IL-12p70	< 300 pg/ml	100-10,000 pg/ml
*MCP-1	< 100 pg/ml	30-3,000 pg/ml
RANTES	< 100 pg/ml	30-3,000 pg/ml
*TGF-b	< 100 pg/ml	30-3,000 pg/ml
TNF-a	<  10 pg/ml	3-1,000 pg/ml

*: for Human Cytokine Ab NewArray Kit, ▲: for Mouse, Rat Cytokine Ab NewArray Kit only.

1. Limits of quantification are conservative estimates based on development of Cytokine antibody NewArray using media diluted antigens. These values are guidelines; actual detection limits may be lower.

2. Standard curve ranges are provided as a guideline. Depending on the application, only part of the suggested curve range may need to be run. Log or half-log dilutions are suggested, if using a narrow standard curve range.

 V.               PROTOCOL

 A.     Blocking

1.     Add 100 µl of 1X Blocking Solution to each well.

2.     Place the slide(s) on an orbital shaker for 30 minutes with gentle agitation.

 B.     Sample Incubation

1.   Remove the 1X Blocking Solution in each well with a pipette and discard and briefly rinse chip twice with 200 µl of 1X Wash Buffer II..

2.   To each well add 15-20 µl of the sample to be analyzed. Samples can be run neat or they can be diluted in 1X Wash Buffer II.

3.   Incubate for 60 min at room temperature.

4.   Remove the sample and let the container upside-down on a lint-free towel to dry.

 C.     Addition of Biotinylated Antibody Cocktail

Decant the samples from each container and wash each chip as follows and wear gloves during the following procedures :

1.   Add 200 µl of 1X Wash Buffer I and incubate for 5 min at room temperature with shaking. Repeat twice (for a total of three washes).

2.   Add 200 µl of 1X Wash Buffer II and incubate for 5 min at room temperature with shaking. Repeat (for a total of two washes). Dab the container upside-down on a lint-free towel to dry.

 

3.   Pipette 20 µl of 1 X Biotin-Conjugated Anti-Cytokine Antibodies Cocktail onto the slide through one of the chamber port holes. Incubate at room temperature for 30mins.

 

D.    Detection

1.   Add 200 µl of 1X Wash Buffer I and incubate for 5 min at room temperature with shaking. Repeat twice (for a total of three washes).

2.   Add 200 µl ml of 1X Wash Buffer II and incubate for 5 min at room temperature with shaking. Repeat (for a total of two washes). Dab the container upside-down on a lint-free towel to dry.

3.   Add 20ul of 1X Streptavidin-Cy5 Conjugate diluted in 1X Blocking Buffer. Incubate at room temperature for 30 minutes.

4.   Add 200 µl of 1X Wash Buffer I and incubate for 5 min at room temperature with shaking. Repeat twice (for a total of three washes).

5.   Add 200 µl ml of 1X Wash Buffer II and incubate for 5 min at room temperature with shaking. Repeat (for a total of two washes). Dab the container upside-down on a lint-free towel to dry.

6.   Image the NewArray™ Cytokine Array Slide (s) using any of the fluorescent imagers currently used for microarray applications.

 

VI.            INTERPRETATION OF RESULTS

 

A.     Mapping the Array

Each cytokine antibody is arrayed in six-repeated, so there should always be a group of six consecutive spots lighting up to a similar level.

 

Table 2: Cytokine Map

This table maps the cytokine antibodies and control as they are oriented within each array on a slide.

 

 

 

 

 

 

 

 

 

 

 

 

*: for Human Cytokine Ab NewArray Kit, ▲: for Mouse, Rat Cytokine Ab NewArray Kit only.

  B.     Averaging hexa-plicated

Each cytokine antibody is arrayed in hexa-plicated to provide better reliability. Using a compatible imaging system’s software, determine the specific signal of each spot (signal-background). Averaging the hexa-plicated’s signal to determine the specific signal for each cytokine on the array. Then determine the standard deviation of this averaged value. The average specific signal for each cytokine +/- the standard deviation is used to compare cytokine expression levels between samples.

 C.     Qualitative Analysis

Average the six-repeated specific signals of the cytokine antibodies on each array being compared. The average specific signal +/- the standard deviation for the cytokine expression in one sample can be compared to the same in other samples to determine if each sample has a different level of cytokine expression. For a more rigorous comparison, perform a test of significance among means（such as a Student’s Test）.

 D.    Quantitative Analysis

As mentioned before, several of the arrays on Cytokine Ab NewArray  slide can be used to generate standard curves. Quantitation of a sample’s cytokine level is possible by comparison to the appropriate standard curve. Furthermore, multiple purified antigens can be combined to generate multiple standard curves within the same arrays on a slide. (A standard curve must be run for each antigen being quantified)

Once a slide is imaged, six-repeated specific signals, representing an antigen of known concentration, can be averaged and plotted, specific signal vs. concentration. All antigen concentrations included in the standard curve are plotted in the same way. A log-log transform of the data should yield a straight line. Then, a log transform of the average specific signal from a sample, allows a comparison to the standard curve to approximate the concentration of cytokine antigen. This process can be performed for each purified antigen included in the arrays to generate multiple standard curves.

 

VII.        FREQUENTLY ASKED ABOUT CYTOKINE Ab NewArray

 Q: What kinds of samples can I analyze? Which species can I analyze?

A: The array can be used with most of the cytokines in cell culture supernates and serum. All the antibodies on our arrays were raised against cytokines. However, some of the arrayed antibodies may also recognize the corresponding mouse and rat proteins partially. 

Q: How much sample is required for the analysis? 

A: 15-20 ml of patient's serum or supernatant of cancer cell line culture medium. Each array is incubated with 0.4-0.8 ng of immobilized antibody. The amount you add may vary depending on the sample. For the majority of sample, we have found that 10ml is sufficient.

 Q: What kind of data do I obtain from the Cytokine Ab NewArray™?

 A: The green or red fluorescent signals from a spot on the array corresponding to the amount of cytokines captured from the sample applied to the array. These values are used to calculate such as the ratio between the intensity and the protein concentration. The advantage of the array format over conventional protein detection assays is the ability to quickly and easily determine how the levels of tens of cytokines have changed, giving you an idea as to which groups of cytokines are being affected.

 Q: What kinds of research can I do by using Cytokine Ab NewArray™ kit?

 A: Cytokines are involved in a lot of disease processes such as inflammation, RA, sepsis, autoimmune disease, asthma, allergy, etc. They play an important role in drug targets, therapeutics, and biomarkers (as below). So, you can do many researches accurately, fast and cost-effectively by using this Cytokine Ab NewArray™ kit.

 

Drug Targets	Therapeutics	Biomarkers
IL-2: Daclizumab for psoriasis	IL-2: Proleukin for melanoma	Inflammatory disorders
IL-8: ABX-IL8 for psoriasis & RA	IFNa: Rebetol for HCV infection	Autoimune disorders
INFg: HuZAF for Crohn’s IBD	G-CSF: Neupogen for neutropenia	Surrogate marker of immunotoxins

 

VIII.     TROUBLESHOOTING GUIDE

 

 IX.            REFERENCE LIST 

l Baraldi-Junkins, etc., Hematol. Oncol. Clin. North Am. 14, 45-61, viii, 2000.

l Huang, R.P., etc., Anal. Biochem. 294, 55-62, 2001.

l Huang, R.P. J. Immunol. Methods, 255, 1-13, 2001.

l Lin, Y., etc., Cancer Res., 62, 2806-2812, 2002.

l Lin, Y., etc., Cancer Letter., 187, 17-24, 2002.

l Ronald L, etc., J. Proteome Research, 1, 233-237, 2002.

l Wang, C. C., etc., J. Proteome Res. 1, 337-343, 2002.

 X.               ORDERING INFORMATION 

Description	Components	Item No.
Cytokine Ab NewArray	4 NewArray™ Cytokine Array Slide	60-10111
	1 X Biotinylated Antibody Cocktail（0.6ml）	60-10012-01
	10X Blocking Buffer（20ml）	60-10001
	10X Wash Buffer I（20ml）	60-10002
	10X Wash Buffer II（20ml）	60-10003
	4 Incubation Grids （Twelve pad chambers）	60-10004

 

XI.            PRICE LIST 

  

l           NewArray  Slide (containing 2 chips each)

l           0.3ml 1 X Biotinylated Anti-Cytokine Antibody Cocktail (for 2 assays)

l           10X Blocking Buffer (10ml)

l           10X Wash Buffer I ( 10ml )

l           10X Wash Buffer II (10ml)

l           2 Incubation Chambers (Twelve-well chambers)