Chicken interleukin 4(IL-4)
FOR RESEARCH USE ONLY
Assay range：3ng/L - 160ng/L
96 determinations
Purpose
This kit allows for the determination of IL-4 concentrations in Chicken serum, cell
culture supernates and other biological fluids
Principle of the assay
The kit assay Chicken IL-4 level in the sample, use Purified Chicken IL-4 antibody to
coat microtiter plate wells, make solid-phase antibody, then add IL-4 to wells, Combined IL-4
antibody which With HRP labeled, become antibody - antigen - enzyme-antibody complex, after
washing Compley, Add TMB substrate solution,TMB substrate becomes blue color At HRP
enzyme-catalyzed, reaction is terminated by the addition of a sulphuric acid solution and the
color change is measured spectrophotometrically at a wavelength of 450 nm. The
concentration of Chicken IL-4 in the samples is then determined by comparing the O.D. of the
samples to the standard curve.
Materials provided with the kit
wash solution
20ml×1bottle
6ml×1 bottle
Stop Solution
6ml×1 bottle
HRP-Conjugate reagent
Standard（320ng/L）
0.5ml×1 bottle
Microelisa stripplate
Sample diluent
12well×8strips
6ml×1 bottle
Standard diluent
Instruction
1.5ml×1bottle
Closure plate
membrane
Chromogen Solution A
Chromogen Solution B
6ml×1 bottle
6ml×1 bottle
Specimen requirements
1. extract as soon as possible after Specimen collection,and according to the relevant
literature, and should be experiment as soon as possible after the extraction. If it can’t,
specimen can be kept in -20 ℃ to preserve, Avoid repeated freeze-thaw cycles.
2. Can’t detect the sample which contain NaN3, because NaN3 inhibits HRP active.
Assay procedure
1. Dilute and add sample:Dilute Original density Standard as follow table:
160ng/L
80ng/L
40ng/L
20ng/L
10ng/L
5 Standard
4 Standard
3 Standard
2 Standard
1 Standard
150μl Original density Standard+150μl Standard diluent
150μl 5 Standard+150μl Standard diluent
150μl 4 Standard+150μl Standard diluent
150μl 3 Standard +150μl Standard diluent
150μl 2 Standard +150μl Standard diluent
2. Add sample: Set blank wells separay (blank comparison wells don’t add sample and
HRP-Conjugate reagent, other each step operation is same). testing sample well. add Sample
dilution 40μl to testing sample well, then add testing sample 10μl (sample final dilution is
5-fold), add sample to wells , don’t touch the well wall as far as possible, and Gently mix.
3. Incubate: After closing plate with Closure plate membrane , incubate for 30 min at 37℃.
4. Configurate liquid: 30-fold(or 20-fold) wash solution diluted 30-fold (or 20-fold) with distilled
water and reserve.
5. Washing: Uncover Closure plate membrane, discard Liquid, dry by swing, add washing
buffer to every well, still for 30s then drain, repeat 5 times, dry by pat.
6. Add enzyme: Add HRP-Conjugate reagent 50μl to each well, except blank well.
7. Incubate: Operation with 3.
8. Washing: Operation with 5.
9. Color: Add Chromogen Solution A 50ul and Chromogen Solution B 50ul to each well, evade
the light preservation for 10 min at 37℃
10. Stop the reaction: Add Stop Solution50μl to each well, Stop the reaction(the blue color
change to yellow color).
11. Assay: take blank well as zero , Read absorbance at 450nm after Adding Stop Solution and
within 15min.
Steps description
2
Standard, Sample diluent
Add Standard, Sample diluent, incubate for 30 min at 37℃.
Wash 5 time,Add HRP-Conjugate reagent, incubate for 30 min at 37℃.
Wash 5 times,Add Chromogen Solution A and B, incubate for 10 min at 37℃.
Add Stopp Solution
Read absorbance at 450nm within 15 min
calculate
Calculate
Take the standard density as the horizontal, the OD value for the vertical ,draw the
standard curve on graph paper, Find out the corresponding density according to the sample
OD value by the Sample curve, multiplied by the dilution multiple, or calculate the straight line
regression equation of the standard curve with the standard density and the OD value ,with the
sample OD value in the equation, calculate the sample density, multiplied by the dilution factor,
