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Rat Follicle-Stimulating
Hormone（FSH）Elisa Kit
（96T）
? This immunoassay kit allows for the in vitro quantitative determination of rat FSH
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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INTRODUCTION
Follicle-stimulating hormone （FSH） is a hormone synthesized and secreted
by gonadotropes in the anterior pituitary gland. FSH regulates the
development, growth, pubertal maturation, and reproductive processes of
the human body. FSH and Luteinizing hormone （LH） act synergistically in
reproduction.
FSH is a glycoprotein. Each monomeric unit is a protein molecule with a
sugar attached to it; two of these make the full, functional protein. Its
structure is similar to those of LH, TSH, and hCG. The protein dimer
contains 2 polypeptide units, labeled alpha and beta subunits. The alpha
subunits of LH, FSH, TSH, and hCG are identical, and contain 92 amino
acids. The beta subunits vary. FSH has a beta subunit of 118 amino acids
（FSHB）, which confers its specific biologic action and is responsible for
interaction with the FSH-receptor. The sugar part of the hormone is
composed of fucose, galactose, mannose, galactosamine, glucosamine,
and sialic acid, the latter being critical for its biologic half-life. The half-life of
FSH is 3-4 hours. Its molecular wt is 30000.
PRINCIPLE OF THE ASSAY
The microtiter plate provided in this kit has been pre-coated with a
goat-anti-rabbit antibody. Standards or samples are then added to the
appropriate microtiter plate wells with a Horseradish Peroxidase
（HRP）-conjugated FSH and anti-FSH antibody and incubated. Then
substrate solution A and B are added to each well. 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 FSH in the samples is then determined by
comparing the O.D. of the samples to the standard curve.
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DETECTION RANGE
0.4 mIU/ml-100 mIU/ml. The standard curve concentrations used for the
ELISA’s were 100 mIU/ml, 30 mIU/ml, 5 mIU/ml, 1 mIU/ml, 0.4 mIU/ml.
SPECIFICITY
This assay recognizes rat FSH. No significant cross-reactivity or
interference was observed.
SENSITIVITY
The minimum detectable dose of rat FSH is typically less than 0.4 mIU/ml.
The sensitivity of this assay, or Lower Limit of Detection （LLD） was defined
as the lowest concentration that could be differentiated from zero.
MATERIALS PROVIDED
Reagent Quantity
Assay plate 1
Standard（S0-S5） 6x 0.5 ml
Antibody 1 x 6 ml
HRP-conjugate 1 x 6 ml
Wash Buffer
1 x 15 ml
（20×concentrate）
Substrate A 1 x 7 ml
Substrate B 1 x 7 ml
Stop Solution 1 x 7 ml
Standard
Standard
0
Standard
1
Standard
2
Standard
3
Standard
4
Standard
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Concentration
（mIU/ml）
0 0.4 1 5 30 100
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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. 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.
REAGENT PREPARATION
1. Bring all reagents and plate to room temperature for at least 30 minutes
before use. Unused wells need store at 2-8°C and avoid sunlight.
2. Wash Buffer If crystals have formed in the concentrate, warm to room
temperature and mix gently until the crystals have compley dissolved.
Dilute 15 ml of Wash Buffer Concentrate into deionized or distilled water
to prepare 300 ml of Wash Buffer.
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.
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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.
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.
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.
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 HRP-conjugate and 50μl of Antibody to each well （not to
Blank well）. Mix well and then incubate for 1 hour at 37°C.
3. Complete remove the liquid. Then 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 Substrate A and 50μl of 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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5. Add 50μl of Stop Solution to each well. If color change does not
appear uniform, gently tap the plate to ensure thorough mixing.
6. Determine the optical density of each well within 10 minutes, using a
microplate reader set to 450 nm.
CALCULATION OF RESULTS
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 X-axis
against the concentration on theY-axis and draw a best fit curve through the
points on the graph. The data may be linearized by plotting the log of the
FSH 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.
? 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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TECHNICAL HINTS
? 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 until added to the plate.
Keep Substrate Solution protected from light. Substrate Solution should
change from colorless 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.