Mouse Oxidized Low-density
Lipoprotein （OxLDL） ELISA Kit 
 
 
 
 
  This immunoassay kit allows for the in vitro quantitative determination of mouse
OxLDL  concentrations  in  cell  culture  supernates,  serum,  plasma  and  other
biological fluids.
  Expiration date      six months from the date of manufacture
  FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES.
 
 
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INTRODUCTION
Low-density  lipoprotein  （LDL）  is  a  type  of  lipoprotein  that
transports  cholesterol  and  triglycerides  from  the  liver  to
peripheral  tissues.  LDL  is  one  of  the  five  major  groups  of
lipoproteins;  these  groups  include  chylomicrons,  very
low-density  lipoprotein  （VLDL）,  intermediate-density  lipoprotein
（IDL）, low-density lipoprotein, and high-density lipoprotein （HDL）,
although  some  alternative  organizational  schemes  have  been
proposed. Like all lipoproteins, LDL enables fats and cholesterol
to move within the water-based solution of the blood stream. LDL
also  regulates  cholesterol  synthesis  at  these  sites.  It  is  used
medically  as  part  of  a  cholesterol  blood  test,  and  since  high
levels  of  LDL  cholesterol  can  signal  medical  problems  like
cardiovascular disease, it is sometimes called "bad cholesterol".
Oxidized  LDL  （Ox-LDL）  is  a  form  of  LDL  that  has  been
bombarded with oxygen to yield free radicals when it enters into
the wall of an artery. Once within the arterial wall, oxidized LDL
promotes atherosclerosis by attracting other cells and chemicals
to  the  site,  causing  inflammation  at  the  site  of  the  artery,  and 
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laying  the  foundation  for  cholesterol  and  other  fats  to  build  up
within the artery.
Under  the  oxidative  stress,  Ox-LDL  may  take  place  in  the
subendothelial space of  the arterial wall, and a small amount of
Ox-LDL may  also  be  released  into  the  circulation. When  "fully
oxidized LDL" enters the circulation in minor quantities, it will be
rapidly cleared by  the  reticuloendothelial system, particularly  in
the  liver,  or  it  will  be  removed  by  the  preexisting  circulating
autoantibodies  to Ox-LDL.  In  contrast,  the  "minimally modified
LDL,"  in which oxidative modification has not been sufficient  to
cause  changes  recognized  by  scavenger  receptors,  can  be
found in circulation.
PRINCIPLE OF THE ASSAY
The microtiter plate provided in this kit has been pre-coated with
an antibody specific  to OxLDL. Standards or samples are  then
added  to  the  appropriate  microtiter  plate  wells  with  a
biotin-conjugated  polyclonal  antibody  preparation  specific  for
OxLDL and Avidin conjugated to Horseradish Peroxidase （HRP） 
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is  added  to  each microplate well  and  incubated.  Then  a  TMB
（3,3',5,5'  tetramethyl-benzidine）  substrate  solution  is  added  to
each  well.  Only  those  wells  that  contain  OxLDL,
biotin-conjugated  antibody  and  enzyme-conjugated  Avidin  will
exhibit  a  change  in  color.  The  enzyme-substrate  reaction  is
terminated by  the addition  of  a  sulphuric  acid  solution  and  the
color  change  is  measured  spectrophotometrically  at  a
wavelength of 450 nm ± 2 nm. The concentration of OxLDL  in
the  samples  is  then  determined  by  comparing  the O.D.  of  the
samples to the standard curve.
DETECTION RANGE
0.078  µmol/ml-5  µmol/ml.  The  standard  curve  concentrations
used for the ELISA’s were 5 µmol/ml, 2.5 µmol/ml, 1.25 µmol/ml,
0.625 µmol/ml, 0.312 µmol/ml, 0.156 µmol/ml, 0.078 µmol/ml.
SPECIFICITY
This assay  recognizes  recombinant and natural mouse OxLDL.
No significant cross-reactivity or interference was observed. 
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SENSITIVITY
The minimum detectable dose of mouse OxLDL is typically less
than 0.0195 µmol/ml.
The sensitivity of  this assay, or Lower Limit of Detection  （LLD）
was  defined  as  the  lowest  protein  concentration  that  could  be
differentiated from zero.
MATERIALS PROVIDED
Reagent      Quantity
Assay plate  1
Standard  2
Sample Diluent      1 x 20 ml
Biotin-antibody Diluent      1 x 10 ml
HRP-avidin Diluent        1 x 10 ml
Biotin-antibody      1 x 120µl
HRP-avidin  1 x 120µl
Wash Buffer      
1 x 20 ml
  （25×concentrate）
TMB Substrate      1 x 10 ml
Stop Solution      1 x 10 ml 
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STORAGE
1. Unopened  test  kits  should  be  stored  at  2-8°C  upon  receipt
and  the microtiter plate should be kept  in a sealed bag. The
test kit may be used throughout the expiration date of the kit,
provided  it  is  stored  as  prescribed  above.  Refer  to  the
package label for the expiration date.
2. Opened test plate should be stored at 2-8°C in the aluminum
foil bag with desiccants to minimize exposure to damp air. The
kits will remain stable until the expiring date shown, provided it
is stored as prescribed above.    
3.  A microtiter  plate  reader with  a  bandwidth  of  10  nm  or  less
and an optical density  range of 0-3 OD or greater at 450nm
wavelength  is  acceptable  for  use  in  absorbance
measurement.
REAGENT PREPARATION
Bring all reagents to room temperature before use.  
1.  Wash  Buffer    If  crystals  have  formed  in  the  concentrate,
warm  up  to  room  temperature  and  mix  gently  until  the
crystals  have  compley  dissolved.  Dilute  20  ml  of Wash
Buffer Concentrate into deionized or distilled water to prepare
500 ml of Wash Buffer. 
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2.  Standard    Centrifuge  the  standard  vial  at  6000-10000rpm
for  30s.  Reconstitute  the  Standard  with  1.0 ml  of  Sample
Diluent.  This  reconstitution  produces  a  stock  solution  of  5
µmol/ml.  Allow  the  standard  to  sit  for  a  minimum  of  15
minutes with gentle agitation prior  to making serial dilutions.
The  undiluted  standard  serves  as  the  high  standard  （5
µmol/ml）. The Sample Diluent serves as  the zero standard
（0 µmol/ml）. Prepare fresh for each assay. Use within 4 hours
and discard after use.
3.  Biotin-antibody    Centrifuge  the vial before opening. Dilute
to  the  working  concentration  using  Biotin-antibody
Diluent（1:100）, respectively.
4.  HRP-avidin    Centrifuge the vial before opening. Dilute to the
working  concentration  using  HRP-avidin  Diluent（1:100）,
respectively.
Precaution: The Stop Solution provided with  this kit  is an acid solution. Wear
eye, hand, face, and clothing protection when using this material.
OTHER SUPPLIES REQUIRED
  Microplate  reader capable of measuring absorbance at 450
nm, with the correction wavelength set at 540 nm or 570 nm. 
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  Pipettes and pipette tips.
  Deionized or distilled water.
  Squirt  bottle,  manifold  dispenser,  or  automated  microplate
washer.
  An incubator which can provide stable incubation conditions
up to 37°C±0.5°C.
SAMPLE COLLECTION AND STORAGE
  Serum    Use  a  serum  separator  tube  （SST）  and  allow
samples  to  clot  for  30 minutes  before  centrifugation  for  15
minutes at 1000 g. Remove serum and assay immediay or
aliquot  and  store  samples  at  -20°C. Centrifuge  the  sample
again  after  thawing  before  the  assay.  Avoid  repeated
freeze-thaw cycles.
  Plasma    Collect plasma using citrate, EDTA, or heparin as
an anticoagulant. Centrifuge for 15 minutes at 1000 g within
30 minutes  of  collection.  Assay  immediay  or  aliquot  and
store  samples  at  -20°C.  Centrifuge  the  sample  again   after
thawing before the assay. Avoid repeated freeze-thaw cycles.
Note: Grossly hemolyzed samples are not suitable for use in this assay. 
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ASSAY PROCEDURE
Bring  all  reagents  and  samples  to  room  temperature  before  use.  It  is
recommended that all samples, standards, and controls be assayed in duplicate.
All  the  reagents  should  be  added  directly  to  the  liquid  level  in  the well.  The
pipette should avoid contacting the inner wall of the well.
1.  Add 100µl of Standard, Blank, or Sample per well. Cover with
the adhesive strip. Incubate for 2 hours at 37°C.  
2.  Remove the liquid of each well, don’t wash.  
3.  Add 100µl of Biotin-antibody working solution to each well.
Incubate  for  1  hour  at  37°C.  Biotin-antibody  working
solution may appear cloudy. Warm up  to  room  temperature
and mix gently until solution appears uniform.
4.  Aspirate  each  well  and  wash,  repeating  the  process  three
times  for a  total of  three washes. Wash: Fill each well with
Wash  Buffer  （200µl）  and  let  it  stand  for  2  minutes,  then
remove  the  liquid  by  flicking  the  plate  over  a  sink.  The
remaining drops are removed by patting the plate on a paper
towel. Complete removal of liquid at each step is essential to
good performance. 
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5.  Add  100µl  of  HRP-avidin  working  solution  to  each  well.
Cover the microtiter plate with a new adhesive strip. Incubate
for 1 hour at 37°C.
6.  Repeat the aspiration and wash five times as step 4.
7.  Add 90µl of TMB Substrate to each well. Incubate for 10-30
minutes  at  37°C.  Keeping  the  plate  away  from  drafts   and
other temperature fluctuations in the dark.  
8.  Add 50µl of Stop Solution  to each well when  the  first  four
wells  containing  the  highest  concentration  of  standards
develop obvious blue color.  If color change does not appear
uniform, gently tap the plate to ensure thorough mixing.
9.  Determine the optical density of each well within 30 minutes,
using a microplate reader set to 450 nm.
CALCULATION OF RESULTS
Using  the  professional  soft  "Curve  Exert  1.3"  to  make  a  standard  curve  is
recommended, which can be downloaded from our web.
Average  the duplicate  readings  for each standard, control, and
sample and subtract  the average zero standard optical density.
Create  a  standard  curve  by  reducing  the  data  using  computer
software capable of generating a  four parameter  logistic  （4-PL） 
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curve-fit. As an alternative, construct a standard curve by plotting
the mean  absorbance  for  each  standard  on  the  y-axis  against
the concentration on the x-axis and draw a best fit curve through
the points on  the graph. The data may be  linearized by plotting
the  log of  the OxLDL concentrations versus  the  log of  the O.D.
and  the best  fit  line can be determined by  regression analysis.
This procedure will produce an adequate but  less precise  fit of
the data.  If samples have been diluted,  the  concentration  read
from the standard curve must be multiplied by the dilution factor.
LIMITATIONS OF THE PROCEDURE
  The kit should not be used beyond the expiration date on the
kit label.
  Do not mix or substitute reagents with those from other lots or
sources.
  It  is  important  that  the  Standard  Diluent  selected  for  the
standard  curve  be  consistent  with  the  samples  being
assayed.
  If samples generate values higher than the highest standard,
dilute the samples with the appropriate Standard Diluent and
repeat the assay. 
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  Any  variation  in  Standard  Diluent,  operator,  pipetting
technique,  washing  technique,  incubation  time  or
temperature, and kit age can cause variation in binding.
  This  assay  is  designed  to  eliminate  interference  by  soluble
receptors,  binding  proteins,  and  other  factors  present  in
biological samples. Until all  factors have been  tested  in  the
Quantikine  Immunoassay,  the  possibility  of  interference
cannot be excluded.
TECHNICAL HINTS
  Centrifuge vials before opening to collect contents.
  When mixing or reconstituting protein solutions, always avoid
foaming.
  To  avoid  cross-contamination,  change  pipette  tips  between
additions of each standard  level, between sample additions,
and between reagent additions. Also, use separate reservoirs
for each reagent.
  When using an automated plate washer, adding a 30 second
soak  period  following  the  addition  of  wash  buffer,  and/or
rotating  the  plate  180  degrees  between  wash  steps  may
improve assay precision. 
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  To ensure accurate results, proper adhesion of plate sealers
during incubation steps is necessary.
  Substrate Solution should remain colorless or light blue until
added  to  the plate. Keep Substrate Solution protected  from
light. Substrate Solution should change from colorless or light
blue to gradations of blue.
  Stop Solution should be added to the plate in the same order
as  the Substrate Solution. The color developed  in  the wells
will turn from blue to yellow upon addition of the Stop Solution.
Wells  that are green  in color  indicate  that  the Stop Solution
has not mixed thoroughly with the Substrate Solution.