Data sheet

Human IL-4 ELISA Kit

Cat. No: EH0002

Introduction Kit Components

Human IL-4 ELISA kit contains all the necessary reagents Item Quantity
required for performing quantitative measurement of IL-4 96- well plate coated 1 Plate
levels from samples including serum, plasma, culture Standard protein lyophilized 1 vial
medium or other biological fluids in a sandwich ELISA Streptavidin -HRP (20µl) 1 vial
format. Detection Antibody, biotinylated (100 µl) 1 vial
Wash Buffer 20X 50 ml
Assay Buffer 10X 50 ml
Substrate/TMB Solution 12 ml
Stop Solution 12 ml
Plate Sealer 2
Sensitivity: < 0.6 ng/ml Reagent Reservoirs 1
Standard Range : 1.56 ng/ml - 50 ng/ml Expiration date: See on the kit label.
Species reactivity Reacts with: Human

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. 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 20X: If crystals have formed in the concentrate, warm to room temperature and mix gently until the crystals
have completely dissolved. To prepare enough Wash Buffer for one plate, add 50 mL of Wash Buffer 20X into deionized or
distilled water to prepare 1L of Wash Buffer 1X.
 Assay Buffer 10X: If crystals have formed in the concentrate, warm to room temperature and mix gently until the crystals
have completely dissolved. To prepare enough Assay Buffer for one plate, add 20 mL of Assay Buffer Concentrate into
deionized or distilled water to prepare 200 mL of Assay Buffer 1X.
 Cytokine Standard: Store lyophilized vials at +4°C. Standard (recombinant protein) should be stored at -20°C or -80°C
(recommended at -80°C) after reconstitution.
 Avoid repeated freeze-thaw cycles of the cytokine standard.
 Standards should be reconstituted with Assay Buffer 1X.
 This reconstitution produces a stock solution
 Recommended standard range: 50 ng/ml to 1.56 ng/ml.

 Detection Antibody is stable at 4ºC until kit expiration date. It is ready for use. Store at –20ºC. Avoid freeze-thaw cycles. For
1 plate dilute 100µl of the vial into 5 mL of Assay Buffer 1X and distribute 50µl in each well.
 Streptavidin Peroxidase stable at 4ºC until expiration date. Centrifuge spin before opening. Dilute Streptavidin-HRP 1:1000
with Assay Buffer 1X just before use. This dilution must be used within 30min after preparation.
 Substrate solution store at 4ºC. It is ready for use. Store in original container, needs to be kept dark.
 Stop solution store at 4ºC. It is ready for use (Precaution: The Stop Solution provided with this kit is an acid solution).
(continued
Canvax Biotech, S.L. C/Astronoma Cecilia Payne. Edif. Canvax. on reverse
14014 Cordoba, page)
Spain.
P: +34 957 348 066
F : +34 957 346 217
www.canvaxbiotech.com
 Data sheet

Canvax Biotech, S.L. C/Astronoma Cecilia Payne. Edif. Canvax. 14014 Cordoba, Spain.
P: +34 957 348 066
F : +34 957 346 217
www.canvaxbiotech.com
 QUICK PROTOCOL
1. Allow the plates to adjust to room temperature (18-25°C) before opening the
bags.

2. Prepare serial dilutions of Standard in Assay Diluent 1X. Dispense 0.1ml dilutions
of Standard in the appropriated wells. Include a blank.

To run the standard curve make a two steps fold dilution ranging from 50 ng/ml to
1.56 ng/ml. Run each test in duplicate. For blank wells dispense 100µl of Assay
Diluent 1X into wells.
3. Prepare desired dilutions of the samples in Assay Diluent 1X. Dispense 0.1ml
dilutions of the samples in the appropriated wells.

4. Cover plate with the adhesive cover slip and incubate for 1hour at room
temperature.

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

6. Transfer 50 µl Detection Antibody to all wells.

7. Cover plate with the adhesive cover slip and incubate for 1 hour at room
temperature.

8. Wash plate three times with wash Buffer 1X as is indicated, step 5.

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

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

11. Wash plate three times in wash Buffer 1X as is indicated, step 5.

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

14. 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.

15. Read absorbance on a spectrophotometer at 450nm.

Analysis:
16. Create a standard curve (standard absorbance on the y-axis, against concentration
standard on the x-axis). Draw a best fit curve.

17. To determine the concentration of human IL-4 for each sample interpolate to the
standard curve. The concentration read from the standard curve must be
multiplied by the dilution factor.

This product is sold for Research or Laboratory Use Only and is not to be used for diagnostic, on humans or for any drug purposes.