1 
Human high mobility group protein B1
（ （（ （HMGB-1） ）） ） 
 
FOR RESEARCH USE ONLY 
 
Assay range： ：： ：8µg/L -160µg/L                                96 determinations 
Purpose 
This  kit  allows  for  the  determination  of HMGB-1  concentrations  in Human  serum, 
cell culture supernates and other biological fluids 
 
Principle of the assay 
The  kit  assay  Human  HMGB-1  level  in  the  sample,  use  Purified  Human  HMGB-1 
antibody to coat microtiter plate wells, make solid-phase antibody,  then add HMGB-1  to wells, 
Combined  HMGB-1  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  HMGB-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 （320µg/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   2 
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: 
160µg/L 
5 Standard  150µl Original density Standard+150µl Standard diluent 
80µg/L 
4 Standard  150µl 5 Standard+150µl Standard diluent 
40µg/L 
3 Standard  150µl 4 Standard+150µl Standard diluent 
20µg/L 
2 Standard  150µl 3 Standard +150µl Standard diluent 
10µg/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.   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 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   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  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