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Human High Mobility Group
protein B1(HMGB-1)ELISA Kit
(96 T)
? This immunoassay kit allows for the in vitro quantitative determination of human
HMGB-1 concentrations in serum, plasma.
? Expiration date six months from the date of manufacture
? FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES.
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PRINCIPLE OF THE ASSAY
The microtiter plate provided in this kit has been pre-coated with
an antibody specific to HMGB-1. Standards or samples are then
added to the appropriate microtiter plate wells with a
biotin-conjugated antibody preparation specific for HMGB-1 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 HMGB-1, 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 HMGB-1 in the samples is then
determined by comparing the O.D. of the samples to the
standard curve.
DETECTION RANGE
78 pg/ml-5000 pg/ml. The standard curve concentrations used
for the ELISA’s were 5000 pg/ml, 2500 pg/ml, 1250 pg/ml,
625pg/ml, 312 pg/ml, 156 pg/ml, 78 pg/ml.
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SPECIFICITY
This assay recognizes human HMGB-1. No significant
cross-reactivity or interference was observed.
SENSITIVITY
The minimum detectable dose of human HMGB-1 is typically
less than 20 pg/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
5000pg/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
(5000pg/ml). The Sample Diluent serves as the zero
standard (0 pg/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 x-axis against
the concentration on the y-axis and draw a best fit curve through
the points on the graph. The data may be linearized by plotting
the log of the HMGB-1 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
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.