Download name and intended use

MATERIALS PROVIDED
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Thyroid Stimulating Hormone
(TSH)
Catalog No. MYM/US-TSH
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For In Vitro Diagnostic Use Only
NAME AND INTENDED USE
The MYM TSH Quantitative MYM/US-TSH is a solid
phase enzyme linked immunsorbent assay. This
test provides quantitative measurement of thyroid
stimulating hormones (TSH) in human serum.
(For in vitro diagnostic use)
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Microwell Strips: Anti-TSH antibodies
coated wells, 8 x 12 strips, 96 wells
Sample Diluent for sample dilution only
(11 mL)
Enzyme Conjugate (11 ml): Anti-TSH
antibody conjugated to horseradish
peroxidase
Solution A (11 mL): containing H2O2.
Solution B (11 mL):
Tetramethybenzidine.
Reference Standard Set (0.75 mL each
vial): Calibrated to 0, 1.0, 5.0, 10, 20,
and 40 uIU/mL in the sample diluent
against with WHO 2nd IRP 80/558.
Stop Solution: 2 N HCl.
Well Holder: For securing individual
wells.
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Microwell Reader
Pipetor with tips for 50 and 100 uL
STORAGE AND STABILITY
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The MYM/US-TSH Quantitative is a solid phase
enzyme-linked immunosorbent assay (ELISA).
The wells are coated with anti-TSH antibodies.
The samples, Standards and Controls are
incubated in the wells with enzyme conjugate
which is another antibody directed toward a
different region of TSH molecules and chemically
conjugated with horseradish peroxidase. Unbound
enzyme conjugate is washed off and the amount
of bound peroxidase is proportional to the
concentration of the TSH present in the Samples,
Standards and Controls. Upon addition of the
substrate and chromogen, the intensity of color
developed is proportional to the concentration of
TSH in the serum.
Store the kit at 2-8C in a refrigerator.
Keep microwells sealed in a dry bag
with desiccants.
The unopened reagents are stable until
expiration of the kit. Solution A and
Solution B should be colorless; if
solutions turn blue, it must be replaced.
Do not expose test reagents to strong
light during storage or usage.
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WARNING AND PRECAUTION
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MYM TSH is designed for in vitro use
only.
The components in this kit are intended
for use as an integral unit. The
components of different lots should not
be mixed.
Handle all reference standards and test
samples as potentially infectious agents.
The standards were found negative for
Hepatitis B, C and HIV antibodies.
However, because no test methods offer
complete assurance of the absence of
the HIV I/II, Hepatitis B Hepatitis C
virus or other infectious agents, these
materials should be handled at the
Biosafety Level 2 as recommended for
any potentially infectious serum or blood
specimen in the Centers for Disease
Control/National Institutes of Health
Manual” Biosafety in Microbiological and
Description
(uIU/ml)
0
1.0
5.0
10
20
40
Pat #1
Pat#2
Pat#3
Absorbance
450 nm
0.002
0.142
0.526
1.011
1.614
3.140
0.083
0.264
1.700
TSH
(uIU/ml)
0.53
2.12
19.82
PREPARATION FOR ASSAY
Before beginning the test, bring all
samples and reagents to room
temperature (20-25C) and gently mix.
Have all reagents and samples ready
before the start of the assay. Once the
test has begun it must be performed
without any interruptions to get the
most reliable and consistent results.
Use new disposable tips for each
specimen.
ASSAY PROCEDURE
1.
PRINCIPLE OF THE ASSAY
Collect blood by venipuncture, allow to clot and
separate the serum by centrifugation at room
temperature. If sera cannot be assayed
immediately, they can be stored at 2-8C for a
week or frozen for up to six months. Avoid
repeated freezing and thawing of serum sample.
MATERIALS REQUIRED BUT NOT PROVIDED
SUMMARY AND EXPLANATION OF TEST
The secretion of TSH from anterior pituitary is
controlled by thyrotropin releasing hormone
(TRH), produced by the hypothalamus. The TSH
acts upon the thyroid gland to stimulate the
production to thyroxine (T4) and triiodothyronine
(T3). The levels of T4 and T3 normally regulate the
secretion of TSH by their negative feedback effect
at the pituitary and possibly at the hypothalamus.
Therefore, the measurement of circulation TSH
levels is an important part of the investigation of
disorders of the hypothalamic /pituitary/thyroid
axis. The MYM TSH Quantitative employs
microtiter ELISA with optimum sensitivity and
specificity for the measurement of this hormone.
SAMPLE COLLECTION AND HANDLING
Well
No.
A1
B1
C1
D1
E1
F1
G1
H1
B1
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Secure the desired number of coated
wells in the holder.
Dispense 50 uL of Standards, Controls
or Serum samples.
Dispense 100 uL of enzyme conjugate to
each wells
Incubate for 60 minutes at room
temperature.
Remove incubation mixture and rinse
the wells 5 times with running tap
water.
Dispense 100 uL of Solution A and 100
uL of Solution B into each well.
Incubate 30 minutes at room
temperature.
Stop reaction by adding 50 uL of 2 N
HCl solution into each well.
Read O.D. at 450 nm with a microwell
reader.
CALCULATION OF RESULTS
Any microwell reader capable of determining at
450 nm may be used. The TSH value of patient is
obtained as follows:
1.
Plot the concentration (X) of each
Reference Standards against its
absorbance (Y) on full logarithmic graph
paper.
2.
Obtain the value of patient TSH by
reference to the Standard Curve. For
example: (This data is for demonstration purposes only and must not be
used in place of data generated for each
assay).
TSH
3.5
3
O.D. 450 nm
1.
Biomedical Laboratories”, 1984. Never
pipet by mouth. Avoid contact with skin.
2.5
2
1.5
1
0.5
0
0
10
20
30
40
50
TSH (uIU/mL)
QUALITY CONTROL
Each laboratory should utilize internal controls at
several levels to monitor assay performance. The
control should be treated as unknown. Results
obtained should be in agreement with the
assigned value of the control. The controls can be
obtained from commercially available sources, but
should not contain sodium azide as preservatives.
LIMITATIONS
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For diagnostic purposes, the TSH values
should be used as an adjunct to other
data available to the physician.
The MYM TSH Quantitative Kit has been
designed so that the high dose hook
effect will not interfere with TSH
determination at levels up to 25
uIU/mL. Samples with TSH levels that
fall above 25 uIU/mL standard should
be diluted in order to obtain a more
accurate value.
EXPECTED VALUES
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It is recommended that each laboratory
determine its own normal and abnormal
range.
As a guide from published literature, the
value for TSH by immunoassay
techniques generally place the adult
euthyroid range below 6 uIU/mL.
Extensive studies have been conducted
on both normal and abnormal patient
samples. The results of which are
summarized below:
Samples
Euthyroid
Hyperthyroid
Hypothyroid
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Hyperthyroid patients show no reaction
to TRH.
N
110
45
20
Range
Upper Limit
Less than
Greater than
uIU/mL
5.5
0.6
5.0
Some studies have indicated a circadian
and episodic variation associated with
TSH secretion. Newborn infants have
substantially higher levels than are
found later in life.
INDICATION FOR QUANTITATIVE ASSAY OF
TSH
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Normal Serum TSH levels of plasma TSH
are generally reported as under 6 uIU
per milliliter, with most assays being
unable to detect levels less thatn 1 uIU
per milliliter.
Primary hypothyroidism in thyroprivic
and goitrous varieties: An elevated TSH
level is the most sensitive screen test
for detecting hypothyroidism. The TSH
level may be high before any clinical or
laboratory test can show a low
circulating level of T4.
Hypothalmic-pituitary hypothyroidism:
The TSH level is undetectable in the
hypothalamic-pituitary hyprothyroidism
minimal elevations of thyroid hormones
in the blood prevent the TSH release
following administration of TRH. In
addition to possible evidence of
intracranial disease hyposecretion of
TSH is accompanied by hyposecretion of
other pituitary hormones. A subnormal
response of the serum TSH to the
administration of TRH confirms the
presence of hypothalamic-pituitary
hypothyroidism.
Thyroid status determination in person
with low TSH level: The level of thyroid
function can be determined by testing
with thyrotropin releasing hormone
(TRH). Hypothyroid patients react to
TRH administration with greater release
of TSH than do normal persons.
PERFORMANCE CHARACTERISTICS
Accuracy
Recovery studies were performed by mixing an
aliquot of pooled serum and TSH Standards. The
TSH values were measured and percentage of
recovery determined.
Initial
Values
(uIU/mL)
2.5
2.5
2.5
25
Expected
Values
(uIU/mL)
6.25
13.75
26.25
34.16
Observed
Values
(uIU/mL)
6
14
28
36
Recovery
(%)
96
102
106
105
Precision
Inter-assay and intra-assay coefficient of variation
were evaluated at three different pooled serum
samples.
N
Mean (uIU/mL)
S.D. (uIU/mL)
C.V. (%)
INTER-ASSAY
Pool A
Pool B
10
10
3.8
13.3
0.24
0.48
6.45
3.63
Pool C
10
45.5
2.01
4.43
N
Mean (uIU/mL)
S.D. (uIU/mL)
C.V. (%)
INTRA-ASSAY
Pool A
Pool B
10
10
3.59
12.47
0.28
0.32
7.71
2.54
Pool C
10
42.50
1.08
2.54
Specificity
The following hormones were tested for crossreactivity of the assay:
Hormone Tested
HCG 400 mIU/mL
LH 200 mIU/mL
FSH 200 mIU/mL
Produce Color Intensity
Equivalent to TSH
<0.20 uIU/mL
<0.25 uIU/mL
<.020 uIU/mL
A linear study was performed to assess the
sensitivity of the assay
Initial
Value
(uIU/mL)
44
Dilution
Folds
1:2
1:4
1:8
1:16
1:32
Expected
Value
(uIU/mL)
22.0
11.0
5.5
2.75
1.38
Observed
Value
(uIU/mL)
22.5
11.5
5.8
3.1
1.7
1:64
1:128
0.69
0.35
0.9
0.5
The sensitivity by this study was less than 1
uIU/mL. The minimal detectable concentration of
TSH is estimated to be 0.5 uIU/mL. A minimal
detectable concentration is defined as that
concentration of TSH that corresponds to the
absorbance that is two standard deviation from
the mean absorbance of 20 serum samples with
zero dose of TSH.
REFERENCE
1.
Buger, H.G. and Y.C. Patel, Thyropin
Releasing Hormone TSH, J. Clin.
Endocrinol and Metab. 6:83-100, 1977
2.
Field, J.B., Pituitary Thyrotropin:
Mechanism of Action in the Thyroid, S.
Werner and S.H. Ingbar. Ed. Harper &
Row. Hagerstwon, MD, 1978
3.
Hamilton, C.R., L.C. Adams and F.
Maloof, Hyperthyroidism Due to
Thyrotropin Producing Pituitary
Chromophobe Adenoma, New England J.
Med. 283, 1077, 1970
4.
Hock F.L., Metabolic Effect of Thyroid
Hormones, In handbook of Physiology,
sec. 7: Endocrinology vol. III Thyroid
M.A. Greer and D.H. Solomon, Ed.
Williams and Wilkins. Baltimore, MD
1972
5.
Ingbar, S.H. and K.A. Woebar, The
Thyroid Gland, in the textbook of
endocrinology R.H. Williams Ed. W.BN.
Suanders & Co. Philadelphia, PA 1981
6.
Lyon, I.C.T. ,Screening for Congenital
Hypothyroidism, three years experience
New Zealand Me. J. 97, 175, 1984
7.
Ornston B.J. ,Clinical Effects of TRH
Release After i.v. and Oral
Administration in Normal Volunteers and
Patients with Tyroid Disease, Frontiers
of Hormone Research 1, 45-75, 1972
8.
Parker, D.C. Pekary, A.E. et al, Effects
of Normal and Reserved Sleepwake
Cycles Upon Nychtohemeral Rhythmicity
of Plasma Thryotropin: Evidence
suggestive of an Inhibitory Influence in
Sleep, J. Clin. Endocrinol. Metab. 43,
318. 1976
9.
Patel, Y.C. and H.G. Burger,Serum
Thyrotroin (TSH) in Pituitary and/or
Hypothalamic Hypothryroidism: Normal
or Elevated Basal Levels and Paradoxical
Responses to Thyrotropin Releasing
Hormone, J. Clin Endocrinol. Metab. 37,
190. 1973
10. Pierce, J.C. , Pituitary Thyrotropin:
Chemistry in the Thyroid, S. Werner and
S.H. Ingbar, Ed. Harper & Row,
Hagerstown. MD, 1978
11. Scanlon, M.F., B.R. Smith et al ThyroidStimulating Hormone: Neuro-regulation
and Clinical Applications, Clin. Sci. Mol
Med. 55 1 & 129, 1978
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