Human Granzyme A
FOR RESEARCH USE ONLY
Assay range：2ng/L -160ng/L 96 determinations
Purpose
This kit allows for the determination of GZMA concentrations in
Human serum, cell culture supernates and other biological fluids
Principle of the assay
The kit assay Human GZMA level in the sample，use Purified Human
GZMA antibody to coat microtiter plate wells, make solid-phase antibody, then
add GZMA to wells, Combined GZMA 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 GZMA 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 Stopp Solution 6ml×1 bottle
2 HRP-Conjugate
reagent 6ml×1 bottle 8
Standard
（320ng/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
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:
160ng/L 5 Standard 150μl Original density Standard+150μl Standard diluent
80ng/L 4 Standard 150μl 5 Standard+150μl Standard diluent
40ng/L 3 Standard 150μl 4 Standard+150μl Standard diluent
20ng/L 2 Standard 150μl 3 Standard +150μl Standard diluent
10ng/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
3
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
4
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
5
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
