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Human Oxidized Low-density
Lipoprotein (OxLDL) ELISA Kit
This immunoassay kit allows for the in vitro quantitative determination of human
OxLDL concentrations in 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
0.156 µmol/ml-10 µmol/ml. The standard curve concentrations
used for the ELISA’s were 10 µmol/ml, 5 µmol/ml, 2.5 µmol/ml,
1.25 µmol/ml, 0.625 µmol/ml, 0.312 µmol/ml, 0.156 µmol/ml.
SPECIFICITY
This assay recognizes recombinant and natural human OxLDL.
No significant cross-reactivity or interference was observed.
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SENSITIVITY
The minimum detectable dose of human OxLDL is typically less
than 0.039 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 completely 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 10
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 (10
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 immediately 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 immediately 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.
