人过氧化物酶（POD）ELISA试剂盒说明书

库存 ：2636

供应商 ：qybio

检测限 ：20pg/ml-1200pg/m

检测方法 ：ELISA

应用 ：科研检测

标记物 ：见说明

样本 ：血清，血浆，组织匀浆等

人过氧化物酶（POD）ELISA试剂盒说明书FOR RESEARCH USE ONLY
Human Peroxidase
Drug Names
Generic Name：Human Peroxidase (POD) ELISA Kit.
Purpose
This kit allows for the determination of POD in Human tissue, cell culture
supernatant and other biological fluids.
Principle of the assay
The kit assay Human POD level in the sample，use Purified Human POD
antibody to coat microtiter plate wells, make solid-phase antibody, then add
POD to wells, Combined POD 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 POD in the samples is then
determined by comparing the O.D. of the samples to the standard curve.5
Materials provided with the kit
Materials provided
with the kit
48determinations 96 determinations
Stora
ge
User manual 1 1
Closure plate
membrane
2 2
Sealed bags 1 1
Microelisa stripplate 1 1 2-8℃
Standard：1800pg/ml 0.5ml×1 bottle 0.5ml×1 bottle 2-8℃
Standard diluent 1.5ml×1 bottle 1.5ml×1 bottle 2-8℃
HRP-Conjugate
reagent
3ml×1 bottle 6ml×1 bottle 2-8℃
Sample diluent 3ml×1 bottle 6ml×1 bottle 2-8℃
Chromogen Solution
A 3ml×1 bottle 6ml×1 bottle 2-8℃
Chromogen Solution
B 3ml×1 bottle 6ml×1 bottle 2-8℃
Stop Solution 3ml×1 bottle 6ml×1 bottle 2-8℃
wash solution
（20ml×20 fold）
×1bottle
（20ml×30 fold）
×1bottle
2-8℃
人过氧化物酶（POD）ELISA试剂盒说明书Specimen requirements
1. cell culture supernatant-detect secretory components, collect sue a
sterile container, centrifugation 20-min at the speed of 2000-3000 r.p.m.
remove supernatant,detect the composition of cells, Dilut cell suspension
with PBS（PH7.2-7.4）, Cell concentration reached 1 million / ml, repeated
freeze-thaw cycles, damage cells and release of intracellular components,
centrifugation 20-min at the speed of 2000-3000 r.p.m. remove supernatant,
If precipitation appeared, Centrifugal again.
2. Tissue samples- After cutting samples, check the weight,add PBS
（PH7.2-7.4）, Rapidly frozen with liquid nitrogen, maintain samples at
2-8℃ after melting,add PBS（PH7.4）, Homogenized by hand or Grinders,
centrifugation 20-min at the speed of 2000-3000 r.p.m. remove
supernatant.6
3. 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.
4. Can’t detect the sample which contain NaN3, because NaN3 inhibits HRP
active.
Assay procedure
1.Dilute and add sample to Standard: set 10 Standard wells on the ELISA
plates coated, add Standard 100μl to the first and the second well, then add
Standard dilution 50μl to the first and the second well, mix; take out 100μl
form the first and the second well then add it to the third and the forth well
separately. then add Standard dilution 50μl to the third and the forth
well ,mix ; then take out 50μl from the third and the forth well discard, add
50μl to the fifth and the sixth well ,then add Standard dilution 50μl to the fifth
and the sixth well, mix ; take out 50μl from the fifth and the sixth well and add
to the seventh and the eighth well, then add Standard dilution 50μl to the
seventh and the eighth well ,mix ; take out 50μl from the seventh and the
eighth well and add to the ninth and the tenth well, add Standard dilution 50μl
to the ninth and the tenth well, mix , take out 50μl from the ninth and the tenth
well discard(add Sample 50μl to each well after Diluting ,(density: 1200 pg/ml，
800 pg/ml ，400 pg/ml，200pg/ml， 100 pg/ml)
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℃.7
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.
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 mins, 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.8
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.
Calculate
人过氧化物酶（POD）ELISA试剂盒说明书Assay range
20pg/ml-1200pg/ml
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
This chart for reference only9
Storage and validity
1．Storage： 2-8℃.
2．validity： six months.