人可溶性髓系
細胞觸發(fā)受體-1（sTREM-1）
elisa試劑盒實驗說明書（英文版）
Human sTREM-1
FOR RESEARCH USE ONLY
供應(yīng)商：上海遠慕生物
Assay range：8pg/ml-240pg/ml 96 determinations
Purpose
This kit allows for the determination of sTREM-1 concentrations in Human serum,cell culture supernates ａnd other biological fluidsPrinciple of the assay
The kit assay Human sTREM-1 level in the sample, use Purified Human sTREM-1antibody to coat microtiter plate wells, make solid-phase antibody, then add sTREM-1 to wells,Combined sTREM-1 antibody which With HRP labeled, become antibody - antigen -enzyme-antibody complex, after washing Completely, Add TMB substrate solution,TMBsubstrate becomes blue color At HRP enzyme-catalyzed, reaction is terminated by the additionof a sulphuric acid solution ａnd the color change is measured spectrophotometrically at awavelength of 450 nm. The concentration of Human sTREM-1 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
（480pg/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 11Closure plate membrane
2
6 Chromogen Solution B 6ml×1 bottle 12 Sealed bags 1
Specimen requirements
2
1. extract as soon as possible after Specimen collection,and according to the relevantliterature, 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:
240pg/ml 5 Standard 150μl Original density Standard+150μl Standard diluent
120pg/ml 4 Standard 150μl 5 Standard+150μl Standard diluent
60pg/ml 3 Standard 150μl 4 Standard+150μl Standard diluent
30pg/ml 2 Standard 150μl 3 Standard +150μl Standard diluent
15pg/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 Sampledilution 40μl to testing sample well, then add testing sample 10μl （sample final dilution is5-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.
3
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℃.
Add Stop Solution
Read absorbance at 450nm within 15 min
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
4
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 inthe room temperature, ELISA plates coated if has not use up after opened, the plate shouldbe stored in Sealed bag.
2. washing buffer will Crystallization separation, it can be heated the water helps dissolvewhen dilute . Washing does not affect the result.
3. add Sample with sampler Each step, And proofread its accuracy frequently, avoids theexperimental 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 firststandard 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 themicrotiter plate reader as a standard.
8. All samples, washing buffer ａnd each kind of reject should according to infective materialprocess.
9. Do not mix reagents with those from other lots.
Storage ａnd validity
1．Storage： 2-8℃.
2．validity： six months
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