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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
....................................................................................................................... 3
3. PROTEIN STRUCTURE ........................................................................................................................... 4
4. GENE ................................................................................................................................................ 4
5. CLINICAL SIGNIFICANCE......................................................................................................................... 4
6. STANDARD RANGE ............................................................................................................................... 4
7. SENSITIVITY ........................................................................................................................................ 4
8. ACCURACY ......................................................................................................................................... 4
9. SPIKE RECOVERY.................................................................................................................................. 4
10. PRECISION ......................................................................................................................................... 5
2. MATERIALS PROVIDED, KIT STORAGE AND EXPIRATION DATE ...........................................5
11. OTHER SUPPLIES REQUIRED ................................................................................................................... 5
12. STORAGE TEMPERATURE AND PREPARATION OF WORKING SOLUTIONS ........................................................... 6
3. DETAILED PROTOCOL .......................................................................................................8
4. PLATE LAYOUT ...............................................................................................................10
5. PRODUCT USE LIMITATION............................................................................................. 11
Table of contents
TABLE OF CONTENTS............................................................................................................2
1. INTRODUCTION ...............................................................................................................3
1. PRINCIPLE .......................................................................................................................................... 3
2. HUMAN INTERLEUKIN-
1. Principle
CvxBio Human IL-6 ELISA kit is a sandwich ELISA for the quantitative measurement of human IL-6 in cell
culture supernatant, serum, plasma and urine. This assay employs an antibody against human IL-6
coated on a 96-well plate. Once IL-6 or standards are bound to the immobilized antibody, the wells are
washed and biotinylated anti-human IL-6 antibody is added. The plates are revealed by the reaction of
the peroxidase of the HRP-streptavidin conjugated.
2.
IL6 is a protein of 185 amino acids glycosylated at positions 73 and 172. It is synthesized as a precursor
protein of 212 amino acids. Monocytes express at least five different molecular forms of IL6 with
molecular masses of 21,5-28 kDa. They mainly differ by post-translational alterations such as
glycosylation and phosphorylation.
IL6 isolated from various cell types shows some microheterogeneity in its N-terminus.
4. Gene
IL6 gene has a length of approximately 5 kb and contains five exons. It is on the p-arm of chromosome 7
(7p21) between the markers D7S135 and D7S370.
The IL6 gene promoter contains many different regulatory elements allowing the induction of
expression by various stimuli, including glucocorticoids and cAMP. The NF-kappa-B binding site is
responsible in non-lymphoid cells for the induction of the IL6 gene expression by IL1 or TNF. In lymphoid
cells, a factor related to the rel oncogene functions as a repressor that prevents the interaction of
transcription factors with the IL6-kappa-B binding site.
5. Clinical significance
6. Standard range
10-3160 pg/ml. The standard range is the range in which determinations of analyte concentration can
be done with precision, accuracy and linearity.
7. Sensitivity
The limit of detection of this assay is 1 pg/ml. It is the lowest concentration that is possible to detect but
not necessarily quantify with precision and accuracy.
8. Accuracy
The standard of this ELISA has been calibrated against an international standard from NIBSC*. One ng of
supplied standard equals 100 units of 89/548 NIBSCstandard.
Please note that the calibration is batch specific.
9. Spike recovery
10. Precision
The intraassay variation is 2.5%(CV). The interassay variation is 3.8%(CV).
*National Institute of Biological Standards and Control, Potters Bar, Hertfordshire EN6
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. Allow the plates to adjust to
room temperature (18-25°C) before opening the bags. Store the remaining strips
in the foil bag containing the desiccant at 4-8°C.
• 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: On arrival all components of the kit, the protein standard, should
be stored at at -20°C. Avoid repeated freeze-thaw cycles of the cytokine standard.
Preparation of standard curve: Prepare a serial dilution of the standard no more
than 30 min prior to the experiment. Dilute the standard stock solution to create a
standard curve ranging from 3.16-10000 pg/ml according to the scheme below.
For the assay background control (0 pg/ml), use only ”Assay Buffer”.
1. Add 0.3ml Wash Buffer 1x per well. Empty wells with aspiration or
invert the plate and tap microwell strips on absorbent paper.
5. Cover plate with the adhesive cover slip and incubate for 1hour at
37ºC.
8. Cover plate with the adhesive cover slip and incubate for 1 hour at
37ºC.
11.Cover plate with the adhesive cover slip and incubate 30 minutes at
room temperature.
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.
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.