Human TNF-α
FOR RESEARCH USE ONLY
Assay range：20 ng/L -400 ng/L 96 determinations
Purpose
This kit allows for the determination of TNF-α concentrations in Human serum, cell
culture supernates and other biological fluids
Principle of the assay
The kit assay Human TNF-α level in the sample, use Purified Human TNF-α antibody to
coat microtiter plate wells, make solid-phase antibody, then add TNF-α to wells, Combined
TNF-α 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 Human TNF-α in the samples is then determined by comparing the O.D.
of the samples to the standard curve.
Materials provided with the kit
1 wash solution 20ml×1bottle 7 Stop Solution 6ml×1 bottle
2 HRP-Conjugate reagent 6ml×1 bottle 8 Standard （800ng/L） 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
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:
400 ng/L
5 Standard 150µl Original density Standard+150µl Standard diluent
200 ng/L
4 Standard 150µl 5 Standard+150µl Standard diluent
100 ng/L
3 Standard 150µl 4 Standard+150µl Standard diluent
50 ng/L
2 Standard 150µl 3 Standard +150µl Standard diluent
25 ng/L
1 Standard 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 to each well, evade the
light preservation for 15 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 30 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,
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 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 and validity
1．Storage： 2-8℃.
2．validity： six months
