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Rabbit Oxidized Lowdensity
Lipoprotein(OxLDL)
ELISA Kit
Catalog No. CSB-E06991Rb
(96 T)
? This immunoassay kit allows for the in vitro quantitative determination of rabbit
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.
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 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 Ox-LDL. Standards or samples are then added
to the appropriate microtiter plate wells with a biotin-conjugated
antibody preparation specific for Ox-LDL and Avidin conjugated to
Horseradish Peroxidase (HRP) 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 Ox-LDL, 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 Ox-LDL in the samples is then determined by
comparing the O.D. of the samples to the standard curve.
DETECTION RANGE
0.08 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, 0.62
μmol/ml, 0.31 μmol/ml, 0.16 μmol/ml, 0.08 μmol/ml.
SPECIFICITY
This assay recognizes recombinant and natural rabbit Ox-LDL. No
significant cross-reactivity or interference was observed.
SENSITIVITY
The minimum detectable dose of rabbit Ox-LDL is typically less
than 0.02 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
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.
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.
? 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.
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.
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)
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 Ox-LDL 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.
? 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.
? 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.
