Rat Interleukin6 (IL-6)ELISA Kit
FOR RESEARCH USE ONLY
INTRODUCTION
Assay range:3pg/ml-120pg/ml 96 determinations
Purpose
This kit allows for the determination of IL-6 concentrations in Rat
serum, cell culture supernates and other biological fluids
PRINCIPLE OF TEST
The kit assay Rat IL-6 level in the sample, use Purified Rat IL-6 antibody
to coat microtiter plate wells, make solid-phase antibody, then add IL-6 to wells,
Combined IL-6 antibody which With HRP labeled, become antibody - antigen -
enzyme-antibody complex, after washing Completely, 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 Rat IL-6 in the samples is then determined by comparing the
O.D. of the samples to the standard curve.
COMPOSITION OF THE KIT
1 wash solution 20ml×1bottle 7 Stop Solution 6ml×1 bottle
2 HRP-Conjugate
reagent 6ml×1 bottle 8
Standard
(240pg/ml) 0.5ml×1 bottle
3 Microelisa stripplate 12well×8strips 9 Standard diluent 1.5ml×1bottle
4 Sample diluent 6ml×1 bottle 10 Instruction 1
5 Chromogen Solution
A 6ml×1 bottle 11 Closure plate
membrane 2
6 Chromogen Solution
B 6ml×1 bottle 12 Sealed bags 1
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Flughafenstra?e 52a 22335 Hamburg DE
2
SAMPLE PREPARATION
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:
120pg/m
l
5 Standard 150μl Original density Standard+150μl Standard diluent
60pg/ml 4 Standard 150μl 5 Standard+150μl Standard diluent
30pg/ml 3 Standard 150μl 4 Standard+150μl Standard diluent
15pg/ml 2 Standard 150μl 3 Standard +150μl Standard diluent
7.5pg/ml 1 Standard 150μl 2 Standard +150μl Standard diluent
2.add sample:Set blank wells separately (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
IBL International GmbH
Flughafenstra?e 52a 22335 Hamburg DE
3
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
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℃.
IBL International GmbH
Flughafenstra?e 52a 22335 Hamburg DE
4
Add Stopp Solution
Read absorbance at 450nm within 15 min
calculate
CALCULATION OF RESULT
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, the result is the sample actual density.
IMPORTANT NOTES
1. The kit takes out from the refrigeration environment should be balanced
15-30 minutes in the room temperature, ELISA plates coated if has not use
up after opened, the plate should be stored in Sealed bag.
2. washing buffer will Crystallization separation, it can be heated the water
helps dissolve when dilute . Washing does not affect the result.
3. add Sample with sampler Each step, And proofread its accuracy frequently,
avoids the experimental error. add sample within 5 min, if the number of
IBL International GmbH
Flughafenstra?e 52a 22335 Hamburg DE
5
sample is much , recommend to use Volley .
4. if the testing material content is excessively higher (The sample OD is
bigger than the first standard well ),please dilute Sample (n-fold), Please
diluente and multiplied by the dilution factor.(×n×5).
5. Closure plate membrane only limits the disposable use, to avoid
cross-contamination.
6. The substrate evade the light preservation.
7. Please according to use instruction strictly, The test result determination
must take the microtiter plate reader as a standard.
8. All samples, washing buffer and each kind of reject should according to
infective material process.
9. Do not mix reagents with those from other lots.
STORAGE CONDITIONS
? The unopened kit shall be stored at [2-8 ℃] .
? For opened kit can be stored at [2-8 ℃] for up to 1 month. If not be used
recently, the standard should be kept in -20 ℃.
? validity: six months
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