Follicle Stimulating Hormone (FSH) ELISA

For the quantitative determination of FSH in human serum

For “In Vitro Diagnostic” use within the United States of America.
This product is for “Research Use Only” outside of the United States of America.

Catalog Number: 11-FSHHU-E01

Size: 96 wells
Version: 9.1 - ALPCO 05Nov13

26-G Keewaydin Drive, Salem, NH 03079 | P: (800) 592-5726 | F: (603) 898-6854 | ts@alpco.com | www.alpco.com
INTENDED USE
For the direct quantitative determination of Follicle Stimulating Hormone by enzyme
immunoassay in human serum.
For in vitro diagnostic use only.

PRINCIPLE OF THE TEST

The principle of the following enzyme immunoassay test follows a typical two-step capture or
„sandwich‟ type assay. The assay makes use of two highly specific monoclonal antibodies: A
monoclonal antibody specific for FSH is immobilized onto the microwell plate and another
monoclonal antibody specific for a different region of FSH is conjugated to horse radish
peroxidase (HRP). FSH from the sample and standards are allowed to bind to the plate,
washed, and subsequently incubated with the HRP conjugate. After a second washing step, the
enzyme substrate is added. The enzymatic reaction is terminated by addition of the stop
solution. The absorbance is measured on a microtiter plate reader. The intensity of the color
formed by the enzymatic reaction is directly proportional to the concentration of FSH in the
sample.
A set of standards is used to plot a standard curve from which the amount of FSH in patient
samples and controls can be directly read.

CLINICAL APPLICATIONS
Human follicle stimulating hormone (FSH) is a glycoprotein hormone produced by the anterior
pituitary gland. There are three other glycoprotein hormones, namely Thyroid Stimulating
Hormone, Luteinizing Hormone (both produced by anterior pituitary gland) and Human
Chorionic Gonadotropin (produced by the placenta) which are structurally similar. Each
hormone has an alpha and beta subunit. The α subunits of each hormone are similar while the
β subunit is specific to each hormone. The α subunits contain 92 amino acids while the β
subunits vary with each hormone. The β subunit of both FSH and LH contain 115 amino acids,
TSH 110 amino acids, and hCG 147 amino acids.
The FSH and LH hormones function differently in females and males. It is to be noted that in
women the growth and maturation of the ovarian follicle is dependent on FSH, while in men both
LH and FSH act on the testes.

PROCEDURAL CAUTIONS AND WARNINGS

1. Users should have a thorough understanding of this protocol for the successful use of this kit.
Reliable performance will only be attained by strict and careful adherence to the instructions
provided.
2. Control materials or serum pools should be included in every run at a high and low level for
assessing the reliability of results.
3. When the use of water is specified for dilution or reconstitution, use deionized or distilled
water.
4. In order to reduce exposure to potentially harmful substances, gloves should be worn when
handling kit reagents and human specimens.
5. All kit reagents and specimens should be brought to room temperature and mixed gently but
thoroughly before use. Avoid repeated freezing and thawing of reagents and specimens.
6. A calibrator curve must be established for every run.
7. The controls should be included in every run and fall within established confidence limits.
8. Improper procedural techniques, imprecise pipetting, incomplete washing as well as improper
reagent storage may be indicated when assay values for the controls do not reflect established
ranges.
9. When reading the microplate, the presence of bubbles in the microwells will affect the optical
densities (ODs). Carefully remove any bubbles before performing the reading step.
10. The substrate solution (TMB) is sensitive to light and should remain colorless if properly
stored. Instability or contamination may be indicated by the development of a blue color, in
which case it should not be used.
11. When dispensing the substrate and stop solution, do not use pipettes in which these liquids
will come into contact with any metal parts.
12. To prevent contamination of reagents, use a new disposable pipette tip for dispensing each
reagent, sample, standard and control.
13. Do not mix various lot numbers of kit components within a test and do not use any
component beyond the expiry date printed on the label.
14. Kit reagents must be regarded as hazardous waste and disposed of according to national
regulations.

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LIMITATIONS
1. All the reagents within the kit are calibrated for the direct determination of FSH in human
serum. The kit is not calibrated for the determination of FSH in saliva, plasma or other
specimens of human or animal origin.
2. Do not use grossly hemolyzed, grossly lipemic, icteric or improperly stored serum.
3. Any samples or control sera containing azide or thimerosal are not compatible with this kit, as
they may lead to false results.
4. Only calibrator A may be used to dilute any high serum samples. The use of any other
reagent may lead to false results.
5. The results obtained with this kit should never be used as the sole basis for clinical diagnosis.
For example, the occurrence of heterophilic antibodies in patients regularly exposed to animals
or animal products has the potential of causing interferences in immunological tests.
Consequently, the clinical diagnosis should include all aspects of a patient‟s background
including the frequency of exposure to animals/products if false results are suspected.
6. Some individuals may have antibodies to mouse protein that can possibly interfere in this
assay. Therefore, the results from any patients who have received preparation of mouse
antibodies for diagnosis or therapy should be interpreted with caution.

SAFETY CAUTIONS AND WARNINGS

POTENTIAL BIOHAZARDOUS MATERIAL
Human serum that may be used in the preparation of the standards and controls has been
tested and found to be non-reactive for Hepatitis B surface antigen and has also been tested for
the presence of antibodies to HCV and Human Immunodeficiency Virus (HIV) and found to be
negative. However no test method can offer complete assurance that HIV, HCV and Hepatitis B
virus or any infectious agents are absent. The reagents should be considered a potential
biohazard and handled with the same precautions as applied to any blood specimen.

CHEMICAL HAZARDS
Avoid contact with reagents containing TMB, hydrogen peroxide and sulfuric acid. If contacted
with any of these reagents, wash with plenty of water. TMB is a suspected carcinogen.

SPECIMEN COLLECTION AND STORAGE

Approximately 0.1 ml of serum is required per duplicate determination. Collect 4-5 ml of blood
into an appropriately labeled tube and allow it to clot. Centrifuge and carefully remove the serum
layer. Store at 4oC for up to 24 hours or at -10oC or lower if the analyses are to be done at a
later date. Consider all human specimens as possible biohazardous materials and take
appropriate precautions when handling.

SPECIMEN PRETREATMENT
This assay is a direct system; no specimen pretreatment is necessary.

REAGENTS AND EQUIPMENT NEEDED BUT NOT PROVIDED

1. Precision pipettes to dispense 25, 50, 100 and 300 μl
2. Disposable pipette tips
3. Distilled or deionized water
4. Plate shaker
5. Microwell plate reader with a filter set at 450nm and an upper OD limit of 3.0 or greater* (see
assay procedure step 13)
6. Centrifuge

REAGENTS PROVIDED
1. Mouse Anti-FSH Antibody Coated Microwell Plate-Break Apart Wells - Ready To Use.
Contents: One 96 well (12x8) monoclonal antibody-coated microwell plate in a resealable
pouch with desiccant.
Storage: Refrigerate at 2-8oC
Stability: 12 months or as indicated on label.

2. Mouse Anti-FSH Antibody-Horseradish Peroxidase (HRP) Conjugate - Requires

Preparation.
Contents: Anti-FSH monoclonal antibody-HRP conjugate in a protein-based buffer with a non-
mercury preservative.
Volume: 300 µl/vial
Storage: Refrigerate at 2-8oC
Stability: 12 months or as indicated on label.
Preparation: Dilute 1:50 in assay buffer before use (eg. 40 µl of HRP in 2 ml of assay buffer). If
the whole plate is to be used dilute 240 µl of HRP in 12 ml of assay buffer. Discard any that is
left over.

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3. FSH Calibrators - Ready To Use.
Contents: Six vials containing FSH in a protein-based buffer with a non-mercury preservative.
Prepared by spiking buffer with a defined quantity of FSH. Calibrated against World Health
Organization (WHO) 1st IS 83/575.
*Listed below are approximate concentrations, please refer to vial labels for exact
concentrations.
Calibrator Concentration Volume
Calibrator 0 IU/L 2.0 ml
A
Calibrator 5 IU/L 0.5 ml
B
Calibrator 10 IU/L 0.5 ml
C
Calibrator 20 IU/L 0.5 ml
D
Calibrator 50 IU/L 0.5 ml
E
Calibrator 100 IU/L 0.5 ml
F
Storage: Refrigerate at 2-8oC
Stability: 12 months in unopened vials or as indicated on label. Once opened, the standards
should be used within 14 days or aliquoted and stored frozen. Avoid multiple freezing and
thawing cycles.

4. Controls - Ready To Use.

Contents: Two vials containing FSH in a protein-based buffer with a non-mercury preservative.
Prepared by spiking serum with defined quantities of FSH. Refer to vial labels for the
acceptable range.
Volume: 0.5 ml/vial
Storage: Refrigerate at 2-8oC
Stability: 12 months in unopened vial or as indicated on label. Once opened, the controls
should be used within 14 days or aliquoted and stored frozen. Avoid multiple freezing and
thawing cycles.

5. Wash Buffer Concentrate - Requires Preparation.

Contents: One bottle containing buffer with a non-ionic detergent and a non-mercury
preservative.
Volume: 50 ml/bottle
Storage: Refrigerate at 2-8oC
Stability: 12 months or as indicated on label.
Preparation: Dilute 1:10 in distilled or deionized water before use. If the whole plate is to be
used dilute 50 ml of the wash buffer concentrate in 450 ml of water.

6. Assay Buffer - Ready To Use.

Contents: One vial containing a protein-based buffer with a non-mercury preservative.
Volume: 25 ml/vial
Storage: Refrigerate at 2-8oC
Stability: 12 months or as indicated on label.

7. TMB Substrate - Ready To Use.

Contents: One bottle containing tetramethylbenzidine and hydrogen peroxide in a non-DMF or
DMSO containing buffer.
Volume: 16 ml/bottle
Storage: Refrigerate at 2-8oC
Stability: 12 months or as indicated on label.

8. Stop Solution - Ready To Use.

Contents: One vial containing 1M sulfuric acid.
Volume: 6 ml/vial
Storage: Refrigerate at 2-8oC
Stability: 12 months or as indicated on label.

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ASSAY PROCEDURE
Specimen Pretreatment: None.
All reagents must reach room temperature before use. Calibrators, controls and specimen
samples should be assayed in duplicate. Once the procedure has been started, all steps should
be completed without interruption.
1. Prepare working solutions of the anti-FSH-HRP conjugate and wash buffer.

2. Remove the required number of microwell strips. Reseal the bag and return any unused
strips to the refrigerator.

3. Pipette 25 µl of each calibrator, control and specimen sample into correspondingly labeled
wells in duplicate.

4. Pipette 100 µl of assay buffer into each well (It is recommended to use a multichannel
pipette).

5. Incubate on a plate shaker (approximately 200rpm) for 30 minutes at room temperature.

6. Wash the wells 3 times with 300 μl of diluted wash buffer per well and tap the plate firmly
against absorbent paper to ensure that it is dry (The use of a washer is recommended).

7. Pipette 100 µl of the conjugate working solution into each well (It is recommended to use a
multichannel pipette).

8. Incubate on a plate shaker (approximately 200 rpm) for 30 minutes at room temperature.

9. Wash the wells again in the same manner as step 6.

10. Pipette 100 µl of TMB substrate into each well at timed intervals.

11. Incubate on a plate shaker for 15-20 minutes at room temperature (or until calibrator F
attains dark blue color for desired OD).

12. Pipette 50 µl of stop solution into each well at the same timed intervals as in step 10.

13. Read the plate on a microwell plate reader at 450 nm within 20 minutes after addition of the
stop solution.

* If the OD exceeds the upper limit of detection or if a 450 nm filter is unavailable, a 405 or 415
nm filter may be substituted. The optical densities will be lower, however, this will not affect the
results of patient/control samples.

CALCULATIONS
1. Calculate the mean optical density of each calibrator duplicate.
2. Calculate the mean optical density of each unknown duplicate.
3. Subtract the mean absorbance value of the “0” calibrator from the mean absorbance values
of the calibrators, controls and serum samples.
4. Draw a calibrator curve on log-log paper with the mean optical densities on the Y-axis and the
calibrator concentrations on the X-axis. If immunoassay software is being used, a 4-parameter
or 5-parameter curve is recommended.
5. Read the values of the unknowns directly off the calibrator curve.
6. If a sample reads more than 100 IU/L then dilute it with calibrator A at a dilution of no more
than 1:8. The result obtained should be multiplied by the dilution factor.

TYPICAL TABULATED DATA

Calibrator OD 1 OD 2 Mean OD Value
(IU/L)
A 0.073 0.071 0.072 0
B 0.218 0.209 0.214 5
C 0.336 0.347 0.342 10
D 0.603 0.601 0.602 20
E 1.433 1.360 1.397 50
F 2.374 2.370 2.372 100
Unknown 0.269 0.263 0.266 7.2

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TYPICAL CALIBRATOR CURVE
Sample curve only. Do not use to calculate results.

10

OD 450nm
1

0.1
5 10 20 50 100

hFSH (IU/L)

PERFORMANCE CHARACTERISTICS

SENSITIVITY
The lower detection limit is calculated from the standard curve by determining the resulting
concentration of the mean OD of Calibrator A (based on 10 replicate analyses) plus 2 SD.
Therefore, the sensitivity of the Direct FSH ELISA kit is 1 IU/L.

SPECIFICITY (CROSS REACTIVITY)

The specificity of the Direct hFSH ELISA kit was determined by measuring the apparent hFSH
value of calibrator A spiked with the following compounds:
Substance Concentration Apparent
Range hFSH
Value
(IU/L)
hCG 1000-50,000 Not
Calibrated IU/L Detected
against WHO
3rd IS 75/537
hLH 5-250 IU/L Not
Calibrated Detected
against WHO
2nd IS 80/552
hTSH 5-250 mIU/L <4.0
Calibrated
against WHO
2nd IS 80/558

INTRA-ASSAY PRECISION
Three samples were assayed ten times each on the same calibrator curve. The results (in IU/L)
are tabulated below:
Sample Mean SD CV%
1 7.41 0.43 5.8
2 48.57 1.70 3.5
3 138.12 4.70 3.4

INTER-ASSAY PRECISION
Three samples were assayed ten times over a period of four weeks. The results (in IU/L) are
tabulated below:
Sample Mean SD CV%
1 7.11 0.24 3.4
2 44.31 2.01 4.5
3 120.63 7.74 7.7

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RECOVERY
Spiked samples were prepared by adding defined amounts of FSH to three patient serum
samples. The results (in IU/L) are tabulated below:
Sample Obs.Result Exp.Result Recovery%
1 5.65 - -
Unspiked 14.75 15.35 96.1
+9.7 56.68 59.15 95.8
+53.5 103.33 112.65 91.7
+107.0
2 17.52 - -
Unspiked 26.21 27.22 96.3
+9.7 70.07 71.02 98.7
+53.5 116.40 124.52 93.5
+107.0
3 58.47 - -
Unspiked 72.71 68.17 106.7
+9.7 114.25 111.97 102.0
+53.5 171.05 165.47 103.4
+107.0

LINEARITY
Three patient serum samples were diluted with calibrator A. The results (in IU/L) are tabulated
below:
Sample Obs.Result Exp.Result Recovery%
1 22.56 - -
1:2 11.89 11.28 105.4
1:4 6.47 5.64 114.7
1:8 3.27 2.82 116.0
2 123.42 - -
1:2 66.80 61.71 108.2
1:4 31.78 30.86 103.0
1:8 17.05 15.43 110.5
3 162.67 - -
1:2 77.93 81.34 95.8
1:4 39.35 40.67 96.8
1:8 21.86 20.33 107.5

HIGH DOSE HOOK EFFECT

The Direct hFSH ELISA kit did not experience any high dose hook effect when tested up to a
FSH concentration of 50,000 IU/L.

REFERENCE VALUES
As for all clinical assays each laboratory should collect data and establish their own range of
expected normal values.
Group Range (IU/L)
Males 1-18
Females
Follicular Stage 2-10
Midcycle Peak 7-20
Luteal Stage 1-10
Postmenopausal 18-150

REFERENCES
1. Butt, W.R. et al., J. Endocrinol. 58:275, 1973.
2. Kjeld, J.M., et., al., Acta Endocrinol. 81:225, 1976.
3. Marshall, J.C., et al., J. Endocrinol. 56:431, 1973.
4. Seth J., et al., Clin. Chim. Acta. 186:67, 1989.
5. Jockenhovel F., et al., J. Clin. Chem. Clin.
Biochem 27:825, 1989.
6. Shome B.,et al.,J. Clin. Endo. Metal. 39:199, 1974.
7. Shome B., et al., J. Clin. Endo. Metal. 39:203, 1974.
8. Labrie F., et al., In Karolinska Symposium on Research method in Reproductive
Endocrinology (Diczfalusy,E,ed) Acta Endocrinol. supl. 180:301, 1973.
9. Knobil, E., Rec. Prog. Horm. Res. 36:53, 1980.

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