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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