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,