CANVAX HUMAN IL-23 ELISA

Manual for cat. nº :

Upon Receipt
Store Kits at +4ºC

PRODUCT MANUAL
Version 3.0
Last updated: July 2012

www.canvaxbiotech.com
1 Canvax Citokine ELISA: Better Tools for the Best Science
Table of contents

TABLE OF CONTENTS............................................................................................................2
1. INTRODUCTION ...............................................................................................................3
1. PRINCIPLE .......................................................................................................................................... 3
2. HUMAN IL-23 .................................................................................................................................... 3
3. PROTEIN STRUCTURE ........................................................................................................................... 3
4. GENE ................................................................................................................................................ 4
5. CLINICAL SIGNIFICANCE......................................................................................................................... 4
6. SENSITIVITY ........................................................................................................................................ 4
7. SPECIFICITY ........................................................................................................................................ 4
2. MATERIALS PROVIDED, KIT STORAGE AND EXPIRATION DATE ...........................................5
8. STORAGE TEMPERATURE AND PREPARATION OF WORKING SOLUTIONS ........................................................... 5
3. DETAILED PROTOCOL .......................................................................................................7
9. CALCULATION OF RESULTS. ................................................................................................................... 8
REFERENCES ........................................................................................................................9

2 Canvax Citokine ELISA: Better Tools for the Best Science

1. INTRODUCTION

1. Principle

CvxBio Human IL-23 ELISA kit is a sandwich ELISA for the quantitative measurement of human IL-23 in
cell culture supernatant, serum, plasma and urine. This assay employs an antibody against human IL-23
coated on a 96-well plate. Once IL-23 or standards are bound to the immobilized antibody, the wells are
washed and biotinylated anti-human IL-17A antibody is added. The plates are revealed by the reaction
of the peroxidase of the HRP-streptavidin conjugated.

2. Human IL-23

Interleukin-23 (IL-23) is a member of the IL-12 family of cytokines with pro-inflammatory properties. Its
ability to potently enhance the expansion of T helper type 17 (Th17) cells indicates the responsibility for
many of the inflammatory autoimmune responses. Emerging data demonstrate that IL-23 is a key
participant in central regulation of the cellular mechanisms involved in inflammation. Both IL-23 and IL-
17 form a new axis through Th17 cells, which has evolved in response to human diseases associated
with immunoactivation and immunopathogeny, including bacterial or viral infections and chronic
inflammation. Interleukin-23 is mainly secreted by activated macrophages and dendritic cells located in
peripheral tissues (skin, intestinal mucosa and lung)

3. Protein Structure

Interleukin-23 is a heterodimer with a 19 000 molecular weight fourfold helical core a subunit (IL-
23p19), disulphide linked to an additional 40 000 molecular weight distinct b subunit (IL-12p40). The p19

3 Canvax Citokine ELISA: Better Tools for the Best Science

expression is produced by antigen-presenting cells as well as by T cells and endothelial cells. The p40 is
primarily restricted to antigen-presenting cells such as monocytes, macrophages and dendritic cells. The
formation of biologically active IL-23 requires the synthesis of both subunits p40 and p19 within the
same cell. Both IL-12 and IL-23 bind to the b1 receptor of T cells and natural killer (NK) cells via their
shared p40 subunit.

4. Gene

The p19 subunit is composed of four exons and three introns; its gene is located on chromosome
12q13.2. However, the p40 subunit consists of eight exons and seven introns, coded by a gene present
on 11q1.3.

5. Clinical significance

• IL-23 is an important part of the inflammatory response against infection.

• Interleukin-23 has been implicated in several autoimmune inflammatory disorders such as colitis,
gastritis, psoriasis and arthritis, and as a novel pro-inflammatory cytokine, with close resemblance to
IL-12.

6. Sensitivity

7. Specificity

4 Canvax Citokine ELISA: Better Tools for the Best Science

2. MATERIALS PROVIDED, KIT STORAGE AND EXPIRATION
DATE
CANVAX HUMAN IL-23 ELISA

Item Quantity Storage

96- well plate coated with coating antibody and blocked non-specific 1 Plate
4ºC
binding sites
Human IL-23 Standard protein lyophilised (1µg) 1 vial 4ºC
Streptavidin -HRP 20µl 4ºC
Detection Antibody, biotinylated 6 ml 4ºC
Wash Buffer 20X 50ml 4ºC
Assay Buffer 25 ml 4ºC
Substrate/TMB Solution 12 ml 4ºC
Stop Solution 12 ml 4ºC
Adhesive cover slip. 2
1

Expiration date: See on the kit label.

8. Storage temperature and preparation of working solutions

Store unopened kit at 4ºC until ready to use. After opening the kit and reconstituting the
reagents, storage following the guidelines below.
• 96-well plate is stable at 4ºC until kit expiration date.
• Wash Buffer is stable at 4ºC until kit expiration date. Add 50ml Wash Buffer 5x to 950ml
deionized water and mix. Store at 4ºC until kit expiration date.
• Assay Buffer. This solution is ready for use. Storage at 4ºC until kit expiration date.
• Cytokine Standard is stable at 4ºC until kit expiration date. Optimized standard curve
range (pg/ml) XXX. Quick-spin before opening. Reconstitute lyophilized antigen standard
with Assay Diluent according to the Cytokine Standard card specifications. Mix until fully
reconstituted. Add a protein carrier for long-term storage, Storage at –80ºC, stable for
several months ? Avoid freeze-thaw cycles

5 Canvax Citokine ELISA: Better Tools for the Best Science

• Detection Antibody is stable at 4ºC until kit expiration date. It is ready for use. Store at –
20ºC as aliquots for ? Avoid freeze-thaw cycles.
• Streptavidin Peroxidase stable at 4ºC until expiration date. Centrifuge spin before
opening. Dilute Streptavidin-HRP 1:1000 with Assay Buffer just before use. This dilution
must be used within 30min after preparation.
• Substrate solution store at 4ºC. It is ready for use.
Substrate Solution should remain colorless until added to the plate. Keep Substrate Solution protected from
light. Substrate Solution should change from colorless to gradations of blue.

• Stop solution store at 4ºC. It is ready for use.

Stop Solution should be added to the plate in the same order as the Substrate Solution. The color
developed in the wells will turn from blue to yellow upon addition of the Stop Solution.
Avoid sodium azide in Buffers because is an inhibitor of peroxidase activity.

6 Canvax Citokine ELISA: Better Tools for the Best Science

3. DETAILED PROTOCOL

1. Add 0.3ml Wash Buffer 1x per well. Empty wells with aspiration or
invert the plate and tap microwell strips on absorbent paper.

2. Use the microwell plate immediately after washing.

3. Prepare serial dilutions of Standard in Assay Diluent .Dispense

0.1ml dilutions of Standard in the appropriated wells. Include a
blank.

4. Prepare desired dilutions of the samples in Assay Diluent. Dispense

0.1ml dilutions of the samples in the appropriated wells.

5. Cover plate with the adhesive cover slip and incubate for 1hour at
37ºC.

6. Wash plate three times with washing Buffer 1x as is indicated, step

1.

7. Transfer 0.1ml Detection Antibody to all wells.

8. Cover plate with the adhesive cover slip and incubate for 1 hour at
37ºC.

9. Wash plate three times with washing Buffer 1x as is indicated, step

1.

10. Transfer 0.1ml diluted Streptavidin Peroxidase into all wells.

11. Cover plate with the adhesive cover slip and incubate 30 minutes
at room temperature.

12. Wash plate five times in washing Buffer 1x as is indicated, step 1.

13. Transfer 0.1ml substrate solution into each well.
14. Incubate at room temperature in the dark and wait to blue colour
development, between 15 and 30 minutes.

15. Add 0.1ml of stop solution to each well. In positive wells will result
a yellow color. It is important that the Stop Solution is spread
quickly and uniformly throughout the microwells.

16. Read absorbance of microwell within 30 minutes on a

7 Canvax Citokine ELISA: Better Tools for the Best Science

 spectrophotometer at 450nm

9. Calculation of Results.

17. Create a standard curve (standard absorbance on the y-axis, against

concentration standard on the x-axis). Draw a best fit curve.

18. To determine the concentration of human cytokine for each sample

interpolate to the standard curve. The concentration read from the standard
curve must be multiplied by the dilution factor.

8 Canvax Citokine ELISA: Better Tools for the Best Science

References
Tang C, Chen S, Qian H, Huang W. Interleukin-23: as a drug target for autoimmune inflammatory
diseases. Immunology. 2012 Feb;135(2):112-24.

Monteleone I, Pallone F, Monteleone G. Interleukin-23 and Th17 cells in the control of gut
inflammation. Mediators Inflamm. 2009; 2009:297645.

9 Canvax Citokine ELISA: Better Tools for the Best Science

PRODUCT USE LIMITATION
This product is developed, designed and sold exclusively for research purposes and in vitro use only. The
product was not tested for use in diagnostics or for drug development, nor is it suitable for
administration to humans or animals. Please refer to www.canvaxbiotech.com for Material Safety Data
Sheet of the product.
The kit should not be used beyond the expiration date on the kit label.

10 Canvax Citokine ELISA: Better Tools for the Best Science