Product Information

Contents: Human IL-23 (p19/p40) ELISA Ready-SET-Go! Kit (with Pre-Coated Plates)
Catalog Number: 88-7239
Sensitivity: 15 pg/ml
Standard Curve Range: 15 pg/ml - 2000 pg/ml
Sizes: 2 pre-coated plates

Available Formats of This Product
Cat. No.	Format	Excite (nm)	Emit (nm)	Reported Applications
88-7239	Human IL-23 (p19/p40) (Interleukin-23, IL23) ELISA Ready-SET-Go! Kit (with Pre-Coated Plates)	N/A	N/A	ELISAkit

Questions? Please consult our answers to frequently asked questions at http://www.ebioscience.com/faq.

Description

This Human IL-23 Ready-SET-Go! ELISA Kit (including pre-coated plates) contains the necessary reagents, standards, buffers and diluents for performing quantitative enzyme-linked immunosorbent assays (ELISA). This ELISA kit is specifically engineered for accurate and precise measurement of human IL-23 protein levels from samples including serum, plasma, and supernatants from cell cultures. The assay demonstrates parallelism in measuring recombinant and native human IL-23 proteins with a standard curve range of 15 pg/ml to 2,000 pg/ml, and assay sensitivity below 15 pg/ml. The assay has been validated by specific detection of significant levels of native human IL-23 protein in supernatants from a variety of different activated dendritic cell populations. The use of a p19-specific capture antibody and a p40-specific detection antibody renders this sandwich ELISA exquisitely specific for human IL-23. IL-12 p40 monomer and IL-12 p70 were run in the assay at 200 ng/ml with no interference or cross-reactivity observed. A panel of 20 unrelated cytokines was also run in the IL-23 ELISA at 100 ng/ml with no cross reactivity observed.

IL-23 is a heterodimeric cytokine composed of the p40 subunit of IL-12 disulfide-linked with a protein p19. p19, like p35 of IL-12, is biologically inactive by itself. IL-23 interacts with IL-12Rbeta1 and an additional, novel beta2-like receptor subunit with STAT4 binding domain, termed IL-23R. IL-23 is secreted by activated mouse and human dendritic cells. Biological activities of mouse IL-23 are distinct from those of mouse IL-12. Mouse IL-23 was found not to induce significant amounts of IFN-g. Mouse IL-23 does induce strong proliferation of memory T cells (but not naïve T cells), whereas IL-12 has no effect on memory cells. Additionally, mouse IL-23 (but not IL-12) can activate mouse memory T cells to produce the proinflammatory cytokine IL-17. Human IL-23 has biological properties which are less distinct from human IL-12; human IL-23 induces proliferation of memory T cells and induces moderate levels of IFN-g production by naïve and memory T cells, as compared to IL-12. IL-23-dependent, IL-17-producing CD4+ T cells (Th-17 cells) have been identified as a unique subset of Th cells that develops along a pathway that is distinct from the Th1- and Th2- cell differentiation pathways. The hallmark effector molecules of Th1 and Th2 cells, e.g., IFN-g and IL-4, have each been found to negatively regulate the generation of these Th-17 cells. More recently, de novo differentiation of Th-17 cells in the absence of IL-23 has been demonstrated by treatment of naïve CD4 cells with TGF-β1 and IL-6.

Components

Pre-coated ELISA Plate, clone eBio473P19
Detection Antibody: Pre-titrated, biotin-conjugated antibody, clone C8.6
Standard: Recombinant cytokine for generating standard curve and calibrating samples
Assay Diluent: 5X concentrated
ELISA Wash Buffer Powder
Detection enzyme: pre-titrated Avidin-HRP
Substrate Solution: Tetramethylbenzidine (TMB) Substrate Solution
Stop Solution: 5 mls of 1X solution per plate
Certificate of Analysis: Lot-specific instructions for dilution of antibodies and standards

Applications Reported

For research use only, not for diagnostic or therapeutic use.

References

Veldhoen, M., et al. 2006. TGF-beta in the context of an inflammatory cytokine milieu supports de novo differentiation of IL-17-producing T cells. Immunity. 24: 179-189.
Harrington, L.E., et al. 2005. Interleukin 17–producing CD4+ effector T cells develop via a lineage distinct from the T helper type 1 and 2 lineages. Nature Immunol. 6: 1123-1132. Brombacher, F., et al. 2003. Novel IL-12 family members shed light on the orchestration of Th1 responses. Trends Immunol. 24: 207-212.
Iwakura, Y., et al. 2006. The IL-23/IL-17 axis in inflammation. J. Clin. Invest. 116: 1218-1222. Oppmann, B., et al. 2000. Novel p19 protein engages IL-12p40 to form a cytokine, IL-23, with biological activities similar as well as distinct from IL-12. Immunity. 13: 715-725.
Aggarwal, S., et al. 2003. IL-23 promotes a distinct CD4 T cell activation state characterized by the production of IL-17. J. Biol. Chem. 278: 1910-1914.

Related Products

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Cat. 88-7176    Human IL-17A (Interleukin-17A, IL17A) ELISA Ready-SET-Go!
Cat. 16-7232    Functional Grade Purified anti-mouse IL-23 (Interleukin-23, IL23) p19 (clone G23-8)
Cat. 88-7234    Mouse IL-23 (p19/p40, IL23) ELISA Ready-SET-Go!
Cat. 88-7274    Mouse IL-27 (p28/EBI3, IL27) ELISA Ready-SET-Go!
Cat. 88-7876    Human IL-17A (Interleukin-17A, IL17A) ELISPOT Ready-Set-Go!
Cat. 88-7879    Human IL-12 (Interleukin-12, IL12) p70 ELISPOT Ready-Set-Go!
Cat. 88-7916    Human IFNg (Interferon-gamma, IFN-g) ELISA Ready-SET-Go! Kit (with Pre-Coated Plates)
Cat. 88-7929    Human IL-12 (Interleukin-12, IL12) p70 ELISA Ready-SET-Go! Kit (with Pre-Coated Plates)
Cat. 88-7976    Human IL-17A (Interleukin-17A, IL17A) ELISA Ready-SET-Go! Kit (with Pre-Coated Plates)
Cat. 34-8129    Carrier-Free Recombinant Human IL-12 (Interleukin-12, IL12) p70
Cat. 34-8179    Carrier-Free Recombinant Human IL-17A (Interleukin-17A, IL17A)
Cat. 34-8239    Carrier-Free Recombinant Human IL-23 (Interleukin-23, IL23)
Cat. 14-8348    Recombinant Human TGFb1 (Transforming Growth Factor beta 1, TGF-beta1, TGF-b1)

Other Materials Needed

Pipettes and pipettors
Refrigerator
96-well ELISA plate reader (microplate spectrophotometer)
ELISA plate washer

Stability

This ELISA kit is guaranteed to perform as specified at least 12 months from date of receipt if stored and handled as instructed according to this datasheet and the Certificate of Analysis, which is included with the reagents.

Storage Instructions for Cytokine Standards

The frozen cytokine standard is already aliquoted at 20 µl per vial. Upon receipt, frozen cytokine standard should be immediately stored at -80°C; stable for at least 12 months. After thawing, quick-spin vial prior to opening. Do not re-aliquot into smaller fractions. These are single use vials. Use one time and discard. For dilution of the standard, please see instructions on the Certificate of Analysis and follow these as written.

Storage Instructions for other kit reagents

Store at 4°C.

IMPORTANT NOTE: Be certain that no sodium azide is present in the solutions used in this assay, as this inhibits HRP enzyme activity.

Time Requirements

4½-hour incubations
1 hour washing and analyzing samples

Experimental Procedure

Using Assay Diluent, dilute standards as noted on the Certificate of Analysis (C of A). Add 100 µl/well of standard to the appropriate wells. Perform 2-fold serial dilutions of the top standards to make the standard curve. Add 100 µl/well of your samples to the appropriate wells. Cover or seal the plate and incubate at room temperature for 2 hours (or overnight at 4°C for maximal sensitivity).
Aspirate wells and wash 5 times with >250 ul / well Wash Buffer (diluted to 1X). Allowing time for soaking (~ 1 minute) during each wash step increases the effectiveness of the washes. Blot plate on absorbent paper to remove any residual buffer.
Add 100 µl/well of detection antibody diluted in 1X Assay Diluent (dilute as noted on C of A). Seal the plate and incubate at room temperature for 1 hour.
Aspirate/wash as in step 2. Repeat for a total of 5 washes.
Add 100 µl/well of Avidin-HRP* diluted in 1X Assay Diluent (dilute as noted on C of A). Seal the plate and incubate at room temperature for 30 minutes.

*Important: Do not include sodium azide in any buffers, as this will inactivate the HRP.
Aspirate and wash as in step 2. In this wash step, soak wells in Wash Buffer for 1 to 2 minutes prior to aspiration. Repeat for a total of 7 washes.
Add 100 µl/well of Substrate Solution to each well. Incubate plate at room temperature for 15 minutes.
Add 50 µl of Stop Solution to each well.
Read plate at 450 nm. If wavelength subtraction is available, subtract the values of 570 nm from those of 450 nm and analyze data.

Ready-SET-Go Cytokine ELISA Kit Buffers:

Assay Diluent (5X concentrate): Dilute 1/5 in dI water.
ELISA Wash Buffer Powder: Reconstitute in 1 L dI water
Substrate Solution: Ready to use (1X); 100 ul per well.
Stop Solution: Ready to use (1X); 50 ul per well

Standard Calibration

The standard of the Ready-SET-Go! is calibrated against NIBSC standards:

Table 1: Table of Standard Calibration
Table of Standard Calibration
Cytokine	ng of eB standard	ng of NIBSC standard	U of NIBSC standard	NIBSC Lot #
hIL-2	1	1.1	14.6	86/564
hIL-4	1	2.2	22	88/656
hIL-5	1	2.2	22	90/586
hIL-6	1	1.7	170	89/548
hIL-10	1	0.8	4	93/722
hIL-12	1	0.8	8	95/544
hIFN-g	1	1.1	22	87/586
hTNF-a	1	0.9	36	87/650
mIL-2	1	3.1	310	93/566
mIL-4	1	3	30	91/656
mIL-6	1	8.5	850	93/730
mIFN-g*	1		4.5	Gg02-901-533
mTNF-a	1	1.7	340	88/532
* Mouse IFN-g is calibrated using NIH standard (Lot Gg02-901-533) and is measured in Units (U)

Table 2: ELISA Troubleshooting Guide
ELISA Troubleshooting Guide
Problem	Possibility	Solution
A. High Background	1. Improper and inefficient washing	1. Improve efficiency of washing. Fill plates completely, soak for 1 minute per wash, as directed
	2. Cross contamination from other specimens or positive control	2. Repeat ELISA, be careful when washing and pipetting
	3. Contaminated substrate	3. Substrate should be colorless
	4. Incorrect dilutions, e.g., conjugate concentration was too high	4. Repeat test using correct dilutions; check with the recommendations of the antibody manufacturer
B. No signal	1. Wrong substrate was used	1. Repeat ELISA, use the correct substrate
	2. Enzyme inhibitor present in buffers; e.g., sodium azide in the washing buffer and Assay Diluent inhibits peroxidase activity	2. Repeat ELISA, make sure your system contains no enzyme inhibitor
C. Very weak signal	1. Improper and inefficient washing	1. Make sure washing procedure is done correctly
	2. Incorrect dilutions of standard	2. Follow recommendations of standard handling exactly as written on the certificate of analysis
	3. Insufficient incubation time	3. Repeat ELISA, follow the protocol carefully for each step’s incubation time
	4. Incorrect storage of reagents	4. Store reagents in the correct temperature, avoid freeze and thaw, avoid using the “frost free” freezer
	5. Wrong filter in ELISA reader was used	5. Use the correct wavelength setting
D. Variation amongst replicates	1. Improper and inefficient washing	1. Make sure washing procedure is done correctly; see certificate of analysis
	2. Poor mixing of samples	2. Mix samples and reagents gently and equilibrate to proper temperature
	3. Plates not clean	3. Plates should be wiped on bottom before measuring absorbance
	4. Reagents have expired	4. Do not use if past expiration date