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小鼠(T4)小鼠甲状腺素(T4)ELISA试剂盒说明书
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Mouse thyroxine （（T4））
（（ ））
ELISA Kit
Catalog No. CSB-E05083m
(96 tests)
This immunoassay kit allows for the in vitro rapid detection of Mouse T4
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
Thyroxine, or 3,5,3’,5’-tetra-iodothyronine (often abbreviated as
T4), a form of thyroid hormones is the major hormone secreted
by the follicular cells of the thyroid gland. Thyroxine is
synthesized via the iodination and covalent bonding of the
phenyl portions of tyrosine residues found in an initial peptide,
thyroglobulin, which is secreted into thyroid granules. These
iodinated diphenyl compounds are cleaved from their peptide
backbone upon being stimulated by thyroid stimulating hormone.
More in the T3 and T4 section of thyroid.
T4 is transported in blood, with 99.95% of the secreted T4 being
protein bound, principally to thyroxine-binding globulin (TBG),
and, to a lesser extent, to transthyretin and serum albumin. T4 is
involved in controlling the rate of metabolic processes in the
body and influencing physical development. Administration of
thyroxine has been shown to significantly increase the
concentration of nerve growth factor in the brains of adult mice.
Thyroxine is a prohormone and a reservoir for the active thyroid
hormone triiodothyronine (T3) which is about four times more
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potent. T4 is converted in the tissues by deiodinases, including
thyroid hormone iodine peroxidase (TPO), to T3. The "D" isomer
is called "Dextrothyroxine" and is used as a lipid modifying agent.
The half-life of thyroxine once released into the blood circulatory
system is about 1 week.
PRINCIPLE OF THE ASSAY
This assay employs the competitive inhibition enzyme
immunoassay technique. A antibody specific to T4 has been
pre-coated onto a microplate. Standards or samples are added
to the appropriate microtiter plate wells with biotin-conjugated T4
and incubated. A competitive inhibition reaction is launched
between T4 (Standards or samples) and Biotin-conjugated T4
with the pre-coated antibody specific for T4. The more amount of
T4 in samples, the less antibody bound by Biotin-conjugated T4.
Then Avidin conjugated to Horseradish Peroxidase (HRP) is
added to each microplate well and incubated. The substrate
solutions are added to the wells, respectively. And the color
develops in opposite to the amount of T4 in the sample. The
color development is stopped and the intensity of the color is
measured.
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DETECTION RANGE
The standard curve concentrations used for the ELISA’s were
320 ng/ml, 160 ng/ml, 80 ng/ml, 40 ng/ml, 20 ng/ml.
SPECIFICITY
This assay recognizes T4. No significant cross-reactivity or
interference was observed.
MATERIALS PROVIDED
Reagent Quantity
Assay plate 1
Standards (S1-S5) 5
HRP-avidin 1 x 6 ml
Conjugate 1 x 6 ml
1 x 15 ml
Wash Buffer
(20×concentrate)
Substrate A 1 x 6 ml
Substrate B 1 x 6 ml
Stop Solution 1 x 6 ml
Standard S1 S2 S3 S4 S5
Concentration(ng/ml) 20 40 80 160 320
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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 with
desiccants to minimize exposure to damp air. The test kit may
be used throughout the expiration date of the kit. Refer to the
package label for the expiration date.
2. Opened test 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.
TECHNICAL HINTS
1. Bring all reagents and plate to room temperature for at least
30 minutes before use. Unused wells need store at 2-8℃and
avoid sunlight.
2. Wash Buffer If crystals have formed in the concentrate,
warm to room temperature and mix gently until the crystals
have completely dissolved. Dilute 15 ml of Wash Buffer
Concentrate into deionized or distilled water to prepare 300 ml
of Wash Buffer.
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3. 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.
4. 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.
5. Substrate Solution should remain colorless until added to the
plate. Keep Substrate Solution protected from light. Substrate
Solution should change from colorless to gradations of blue.
6. 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.
Precaution: The Stop Solution provided with this kit is an acid solution. Wear
eye, hand, face, and clothing protection when using this material.
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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.
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 x g. Remove serum and assay immediately
or aliquot and store samples at -20° C. Avoid repea ted
freeze-thaw cycles.
Plasma Collect plasma using citrate, EDTA, or heparin as
an anticoagulant. Centrifuge for 15 minutes at 1000 x g within
30 minutes of collection. Assay immediately or aliquot and
store samples at -20° C. 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.
1. Set a Blank well without any solution. Add 50μl of Standard
or Sample per well. Standard need test in duplicate.
2. Add 50μl of Conjugate to each well (not to Blank well), Mix
well and then incubate for 1 hour at 37°C.
3. Fill each well with Wash Buffer (about 200μl), stay for 10
seconds and Spinning. Repeat the process for a total of
three washes. Complete removal of liquid at each step is
essential to good performance. After the last wash, remove
any remaining Wash Buffer by aspirating or decanting.
Invert the plate and blot it against clean paper towels.
4. Add 50μl of HRP-avidin to each well. Incubate for 30mins
at 37°C.
5. Repeat the aspiration and wash five times as step 4.
6. Add 50μl of Substrate A and Substrate B to each well, mix
well. Incubate for 15 minutes at 37°C. Keeping the plate
away from drafts and other temperature fluctuations in the
dark.
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7. Add 50μl of Stop Solution to each well.
8. Determine the optical density of each well within 10 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, Blank, and
sample and subtract the optical density of Blank. 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 T4 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.
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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 Calibrator 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 Calibrator 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
Immunoassay, the possibility of interference cannot be
excluded.
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