FOR INFORMATIONAL USE ONLY ? DO NOT USE FOR PERFORMING ASSAY ?

REFER TO MOST CURRENT PACKAGE INSERT ACCOMPANYING TEST KIT

THYROID STIMULATING HORMONE (TSH) ENZYME IMMUNOASSAY TEST

KIT
Catalog Number: BC-1001

BioCheck, Inc. PRINCIPLE OF THE ASSAY

The BioCheck TSH ELISA test is based on the principle of a solid
837 Cowan Rd. phase enzyme-linked immunosorbent assay.9,10 The assay system
Burlingame, CA 94010 utilizes a unique monoclonal antibody directed against a distinct
antigenic determinant on the intact TSH molecule. Mouse monoclonal
Enzyme Immunoassay for the Quantitative anti-TSH antibody is used for solid phase immobilization (microtiter
Determination of Thyroid Stimulating wells), and goat anti-TSH antibody is in the antibody-enzyme
(horseradish peroxidase) conjugate solution. The test sample is
Hormone (TSH) in Human Serum allowed to react simultaneously with the two antibodies, resulting in the
TSH molecules being sandwiched between the solid phase and
FOR IN VITRO DIAGNOSTIC USE enzyme-linked antibodies. After a 60-minute or overnight incubation at
Store at 2 to 8°C. room temperature, the solid phase is washed with water to remove
unbound labeled antibodies. A solution of 3,3’,5,5’-
PROPRIETARY AND COMMON NAMES Tetramethylbenzidine (TMB) is added and incubated for 20 minutes,
BioCheck TSH Enzyme Immunoassay resulting in the development of a blue color. The color development is
stopped with the addition of 3N HCl, and the resulting yellow color is
INTENDED USE measured spectrophotometrically at 450 nm. The concentration of
For the quantitative determination of thyroid stimulating hormone (TSH) TSH is directly proportional to the color intensity of the test sample.
concentration in human serum. The assay is useful in the diagnosis of
thyroid or pituitary disorders. REAGENTS AND MATERIALS PROVIDED
1. Antibody-Coated Wells (1 plate, 96 wells)
INTRODUCTION Microtiter wells coated with mouse monoclonal anti-TSH.
The determination of serum or plasma levels of thyroid stimulating
2. Enzyme Conjugate Reagent (1 dropper bottle, 13 mL)
hormone (TSH or thyrotropin) is recognized as a sensitive method in
Contains goat anti-TSH conjugated to horseradish peroxidase.
the diagnosis of primary and secondary hypothyroidism. 1 TSH is
secreted by the anterior lobe of the pituitary gland and induces the 3. Reference Standard Set (1 mL/vial)
production and release of thyroxine (T4) and triiodothyronine (T3) Contains 0, 0.5, 2.0, 5.0, 10.0 and 25.0 µIU/mL (WHO, 2nd IRP,
from the thyroid gland.2 It is a glycoprotein with a molecular weight of 80/558) TSH in equine serum with preservatives. Lyophilized.
approximately 28,000 daltons, consisting of two chemically different See instructions for reconstitution under Reagent Preparation.
subunits, alpha and beta.3 4. TMB Reagent (1 bottle, 11 mL)
Although the concentration of TSH in the blood is extremely low, it Contains 3, 3’, 5, 5’ tetramethylbenzidine (TMB) stabilized in
is essential for the maintenance of normal thyroid function. The release buffer solution.
of TSH is regulated by a TSH-releasing hormone (TRH) produced by 5. Stop Solution (1N HCl) (1 bottle, 11 mL)
the hypothalamus. The levels of TSH and TRH are inversely related Contains diluted hydrochloric acid.
to the level of thyroid hormone. When there is a high level of thyroid
hormone in the blood, less TRH is released by the hypothalamus, so MATERIALS REQUIRED BUT NOT PROVIDED
less TSH is secreted by the pituitary. The opposite action will occur
1. Distilled or deionized water
when there is decreased thyroid hormone in the blood. This process
2. Precision pipettes: 50 µL, 100 µL, 200 µL, and 1 mL
is known as a negative feedback mechanism and is responsible for
3. Disposable pipette tips
maintaining the proper blood levels of these hormones.4,5
4. Microtiter well reader capable of reading absorbance at 450nm.
TSH and the pituitary glycoproteins: luteinizing hormone (LH),
5. Orbital motion microtiter well shaker, capable of shaking at a
follicle-stimulating hormone (FSH), and human chorionic gonadotropin
speed of 175±25 RPM
(hCG), have identical alpha chains.2 The beta chains are distinct but
6. Absorbent paper
do contain regions with identical amino acid sequences. These regions
7. Graph paper (semi-log, etc.)
of homology can cause considerable cross-reactivity with some
8. Quality control material (e.g., BioRad Lyphochek Control sera)
polyclonal TSH antisera. The use of a monoclonal antibody in
BioCheck’s TSH ELISA test eliminates this interference, which could
result in falsely elevated TSH values in either menopausal or pregnant
females, a population whose evaluation of thyroid status is clinically
significant. 6,7,8
 PROCEDURAL NOTES
WARNINGS AND PRECAUTIONS 1. Manual Pipetting: It is recommended that no more than 32 wells
1. CAUTION: Test methods are not available which can offer be used for each assay run. Pipetting of all standards, samples,
complete assurance that Hepatitis B virus, Human and controls should be completed within 3 minutes.
Immunodeficiency Virus (HIV/HTLV-III/LAV), or other infectious 2. Automated Pipetting: A full plate of 96 wells may be used in each
agents are absent from the reagents in this kit. Therefore, all assay run. However, it is recommended that pipetting of all
human blood products, including serum samples, should be standards, samples, and controls be completed within 3 minutes.
considered potentially infectious. Handling should be as defined 3. All standards, samples, and controls should be run in duplicate
by an appropriate national biohazard safety guideline or concurrently so that all conditions of testing are the same.
regulation, where it exists.11 4. It is recommended that the wells be read within 15 minutes
2. Avoid contact with 1N HCl. It may cause skin irritation and burns. following addition of Stop Solution.
If contact occurs, wash with copious amounts of water and seek
medical attention if irritation persists. ASSAY PROCEDURE
3. Do not use reagents after expiration date and do not mix or use 1. Secure the desired number of coated wells in the holder.
components from kits with different lot numbers. 2. Dispense 100µl of standards, specimens, and controls into
4. Replace caps on reagents immediately. Do not switch caps. appropriate wells.
5. Solutions containing additives or preservatives, such as sodium 3. Dispense 100µl of Enzyme Conjugate Reagent into each well.
azide, should not be used in the enzyme reaction. 4. Thoroughly mix for 30 seconds. It is very important to have
6. Do not pipette reagents by mouth. complete mixing.
7. For in vitro diagnostic use. 5. Incubate at room temperature (18-25°C) for 60 minutes (1 hour).
6. Remove the incubation mixture by flicking plate contents into a
STORAGE CONDITIONS waste container.
1. Store the unopened kit at 2-8°C upon receipt and when it is not in 7. Rinse and flick the microtiter wells 5 times with distilled or
use, until the expiration shown on the kit label. Refer to the dionized water. (Please do not use tap water.)
package label for the expiration date. 8. Strike the wells sharply onto absorbent paper or paper towels to
2. Keep microtiter plate in a sealed bag with desiccant to minimize remove all residual water droplets.
exposure to damp air. 9. Dispense 100µl of TMB Reagent into each well. Gently mix for 5
seconds.
INSTRUMENTATION 10. Incubate at room temperature, for 20 minutes.
A microtiter well reader with a bandwidth of 10nm or less and an 11. Stop the reaction by adding 100µl of Stop Solution to each well.
optical density range of 0 to 2 OD or greater at 450 nm 12. Gently mix for 30 seconds. Ensure that all of the blue color
wavelength is acceptable for absorbance measurement. changes completely to yellow.
13. Read absorbance at 450nm with a microtiter plate reader within
SPECIMEN COLLECTION AND PREPARATION 15 minutes.
1. Serum should be prepared from a whole blood specimen
obtained by acceptable medical techniques. This kit is for use with CALCULATION OF RESULTS
serum samples without additives only. Avoid grossly hemolytic, 1. Calculate the mean absorbance value (OD 450) for each set of
lipemic, or turbid samples. reference standards, controls and samples.
2. Specimens should be capped and may be stored for up to 48 2. Construct a standard curve by plotting the mean absorbance
hours at 2-8°C prior to assaying. Specimens held for a longer obtained for each reference standard against its concentration in
time should be frozen only once at -20°C prior to assay. Thawed µIU/mL, with absorbance on the vertical (y) axis and
samples should be inverted several times prior to testing. concentration on the horizontal (x) axis.
3. Using the mean absorbance value for each sample, determine
REAGENT PREPARATION the corresponding concentration of TSH in µlU/ml from the
1. All reagents should be allowed to reach room temperature (18-25 standard curve. Depending on experience and/or the availability
°C) before use. of computer capability, other methods of data reduction may be
2. All reagents should be mixed by gentle inversion or swirling prior employed.
to use. Do not induce foaming. 4. Any diluted samples must be further corrected by the appropriate
3. Reconstitute each lyophilized standard with 1.0mL dH20. Allow dilution factor.
the reconstituted material to stand for at least 20 minutes.
Reconstituted standards should be stored sealed at 2-8°C, and
EXAMPLE OF STANDARD CURVE
are stable at 2-8°C for at least 30 days.
Results of a typical standard run of the assay are shown below. The
4. Specimens expected to have a TSH concentration greater thatn
standard curve is for illustration only, and should not be used to
25µIU/mL should be diluted 1:10 with TSH-free serum (Zero
calculate unknowns. The standard curve covers a dynamic range
calibrator).
from 0 to 25 µIU/mL.

2
 The absorbance (450nm) value can be varied due to incubation at 1. Accuracy
different room temperatures in different laboratories. BioCheck, Inc.’s TSH ELISA has been compared to DPC’s
Immulite 2000 3rd Generation TSH test. The methods are similar
in that they are both used for the quantitative determination of TSH
TSH (µIU/mL) Absorbance (450nm) in human serum, and both use TSH calibrated and labeled in
0 0.012 µIU/ml standardized to the WHO hTSH (2nd IRP, 80/558).
0.5 0.112
2.0 0.409 A study was conducted using 39 euthyroid and 20 hypothyroid
5.0 0.979 patient samples (as determined by hospital laboratory analysis).
10.0 1.778 The range of samples tested was 0.4 µIU/mL to 63 µIU/mL. The
25.0 3.296 comparison demonstrated good correlation with another
commercial kit as shown below:

N = 59
3.5
Correlation coefficient = 0.994
3 Slope = 1.027
Intercept = 0.787
2.5 Biocheck Mean = 8.39 µIU/mL
Absorbance (450nm)

DPC Mean = 9.41 µIU/ml

2

1.5 Another 77 hyperthyroid patient samples also correlated well with

the DPC test kit:
1
# Samples with [TSH] ≤ 0.2 µIU/mL
0.5 DPC Kit N = 77
BioCheck Kit N = 77
0
0 5 10 15 20 25
BioCheck TSH (µIU/mL) 2. Sensitivity
At TSH concentrations of 0.1 µIU/mL and 0.2 µIU/mL, the
interassay CVs were determined to be 11.4% and 7.9%,
respectively.
EXPECTED VALUES
Each laboratory should establish its own normal range based on 3. Precision
patient population. Differences in assay technique and the use of a. Intra-Assay Precision
various standards may affect results. The results provided below are Within-run precision was determined by replicate
based on 38 normal and 77 hyperthyroid blood specimens. The determinations of four different serum samples in one assay.
ranges were determined from the mean ± 2SD (µIU/mL TSH). These Within-assay variability is shown below:
values may differ from other published data.
Serum Sample 1 2 3 4
Normal Hyperthyroid Number of Replicates 28 26 26 26
N 38 77 Mean TSH (µIU/ml) 0.62 1.51 15.48 26.14
Mean TSH (µIU/mL) 1.44 < 0.2 Standard Deviation 0.03 0.09 0.39 0.86
Range 0.3 - 8.1 < 0.2 Coefficient of Variation 4.6% 5.7% 2.5% 3.3%
(%)

NORMAL REFERENCE RANGE b. Inter-Assay Precision

The reference ranges provided below are based on Tietz.12 Between-run precision was determined by replicate
measurements of four different serum samples over a series
TSH (µU/mL) of individually calibrated assays as shown below:
Adults
Serum Sample 1 2 3 4
21-54 years 0.4-4.2
Number of Replicates 30 24 24 24
55-87 years 0.5-8.9
Mean TSH (µIU/ml) 0.64 1.46 15.38 25.26
Pregnancy
1st Trimester 0.3-4.5 Standard Deviation 0.05 0.10 0.87 1.75
2nd Trimester 0.5-4.6 Coefficient of Variation 7.6% 7.1% 5.7% 8.9%
3rd Trimester 0.8-5.2 (%)

PERFORMANCE CHARACTERISTICS
3
 PERFORMANCE CHARACTERISTICS 65/217) 50 ng/mL 0
200 ng/mL <0.2
4. Recovery and Linearity Studies
a. Recovery 5. Hook Effect
Various patient serum samples of known TSH levels were No hook effect is observed in this assay at TSH concentrations up
combined and assayed in duplicate. The mean recovery to 1,000 µIU/mL.
was 98.9%.
QUALITY CONTROL
Good laboratory practice requires that low, mediumm and high quality
Expected Concentration Observed %
control specimens (controls) be run with each calibration curve to
(µIU/ml) Concentration (µ/ml) Recovery
verify assay performance. To assure proper performance, a
23.94 22.92 95.7%
11.75 11.01 93.6%
statistically significant number of controls should be assayed to establish
5.46 5.53 101.3% mean values and acceptable ranges. Controls containing sodium
2.76 3.04 110.1% azide should not be used.
1.48 1.39 93.9%
STANDARDIZATION
b. Linearity The TSH Reference Standards are calibrated against the World Health
Two patient samples were serially diluted with zero standard Organization’s Second International Reference Preparation of hTSH
to determine linearity. The mean recovery was 94.6%. 2nd IRP-80/558).
# Dilution Expected Observed
Conc. Conc. % Expected LIMITATIONS OF THE PROCEDURE
(µIU/ml) (µIU/ml) 1. Reliable and reproducible results will be obtained when the assay
1. Undiluted 48.39 procedure is carried out with a complete understanding of the
1:2 24.42 22.43 91.9% package insert instructions and with adherence to good
1:4 11.21 10.45 93.2% laboratory practice.
1:8 5.60 5.13 91.6% 2. The results obtained from the use of this kit should be used only
1:16 2.80 2.87 102.5% as an adjunct to other diagnostic procedures and information
1:32 1.40 1.30 92.9% available to the physician.
Average = 94.4% 3. Serum samples demonstrating gross lipemia, gross hemolysis, or
2. Undiluted 36.42 turbidity should not be used with this test.
1:2 18.21 18.10 99.4% 4. The wash procedure is critical. Insufficient washing will result in
1:4 9.10 8.48 93.2% poor precision and falsely elevated absorbance readings.
1:8 4.55 4.28 94.1% 5. The BioCheck TSH assay has not been tested on newborns, and
1:16 2.27 2.22 97.8% is not for use in screening newborns.
1:32 1.13 1.01 89.4%
Average = 94.8% REFERENCES
1. Burger, H. G., Patel, Y. C., Thyrotropin releasing hormone-TSH
6. Specificity Clinic. Endocrinol. and Metab., 6, 831-00(1977).
The following hormones were tested for cross-reactivity: 2. Ezrin, C., The Thyroid, S. C. Werner and S. H. lngbar (eds.),
Harper and Row, Hagerstown, MD, 9, 174-178 (1978).
HORMONE TESTED CONCENTRATION PRODUCED INTENSITY 3. Pierce, J. G., Endocrinology, 89, 1331-1344 (1971).
EQUIVALENT TO TSH 4. Berger, S. and Quinn, J. L., Fund. Clin. Chem., N. W. Tietz (ed.),
(µIUmL)
W. B. Saunders Co., Phila., PA 14, 824-848 (1976).
HCG - (WHO 2nd IS 100 mIU/mL 0
5. Utiger, R. D., The Thyroid, S.C. Werner and S. H. Ingbar (eds.),
61/6) 600 mIU/mL 0
Harper and Row, Hagerstown, MD, 9, 196-205 (1978).
3,500 mIU/mL 0
6. Soos, M. and Siddle, K., J. Immun. Methods, 51, 57-68 (1982).
10,000 mIU/mL 0
200,000 mIU/mL 0
7. Wada, H. G., Danisch, R. J., Baxter, S. R., et al, Clin. Chem.,28,
FSH - (WHO 2nd IRP- 20 mIU/mL 0
1862-1866 (1982).
HMG) 100 mIU/mL 0 8. Snyder, P. J. & Utiger, R. D., J. Clin. Endo. Metab., 34, (1972).
200 mIU/mL <0.2 9. Engall, E., Methods in Enzymology, Vol. 70, VanVunakis, H. &
LH - (WHO 1st IRP 75 mIU/mL 0 Langone, J.J. (eds.), Acad. Press, NY, 419-492(1980).
68/40) 150 mIU/mL 0 10. Uotila, M., Ruoslahti, E. and Engvall, E., J. lmmunol. Methods, 42,
300 mIU/mL <0.2 11-15 (1981).
Prolactin - (WHO 1st 10 ng/mL 0 11. USA Center for Disease Control/National Institute of Health
IRP 75/504) 50 ng/mL 0 Manual, “Biosafety in Microbiological and Biomedical
200 ng/mL <0.2 Laboratories”, 1984.
hGH - (WHO 1st IRP 10 ng/mL 0

4
12. Clinical Guide to Laboratory Tests. Ed. N.W. Tietz, 3rd Ed., W.B.
Saunders Company, Philadelphia, PA 19106, 1995.
TECHNICAL CONSULTATION
Call or Write: BioCheck, Inc.
837 Cowan Road
Burlingame, CA 94010
Tel: (650) 259-8425 Fax: (650) 259-8427

Revision Date 030602