Download A brief review of rat thyroid-stimulating hormone (TSH) and

A brief review of rat thyroid-stimulating hormone (TSH) and
introduction to Shibayagi’s rat TSH ELISA KIT
Katsumi WAKABAYASHI, Ph.D.
Prof. emer. Gunma University
Technical consultant, Shibayagi Co. Ltd.
What is TSH?
TSH is produced and secreted by basophilic cells called “thyrotrophs” in the anterior
pituitary gland of vertebrates from fishes to mammals. TSH is also called thyrotropic
hormone or thyrotropin.
TSH acts on thyroid gland, adipose tissue, eye, and so on. TSH receptor penetrates
the cell membrane 7 times and is coupled with G-protein-PKA system.
TSH increases the release of thyroid hormone, and causes morphological changes in
thyroid tissue, and promotes biosynthesis of the thyroid hormones through
enhancement of inorganic iodide uptake, iodination of tyrosine in thyroglobulin, and
changes of iodotyrosine to thyroid hormones. TSH promotes metabolism of glucose,
phospholipids, and nucleic acids in the thyroid.
In the adipose tissue, TSH enhances glucose uptake, degradation of lipids, and
oxygen consumption.
TSH and TSH metabolites bind to cell membrane of retroorbital tissue and increases
adenylate cyclase activity, causing exophthalmos.
The most characteristic syndrome of TSH deficiency is the reduction of the thyroid
function, in other words, decreases in thyroid hormone production and secretion.
Diseases showing low blood TSH levels: Hyperthyroidism ( Basedow or Graves disease,
Plummer
disease),
panhypopituitarism （ Simmonds
Syndrome ） ，
partial
hypopituitarism， isolated TSH deficiency, Sheehan’s syndrome, etc.
Diseases showing high blood TSH levels: Pituitary tumor, ectopic TSH-producing tumor,
hypothyroidism (cretinism, iodine deficiency), thyroid hormone refractoriness, etc.
Regulation of TSH secretion
Factors for enhancement of secretion
TRH
(Thyrotropin
releasing
hormone):
TRH,
pyroglutamylhistidylprolinamide,
originated in the hypothalamus, directly stimulates TSH secretion. TRH, at a
concentration of 10pg/ml, increased TSH release from rat anterior pituitary in vitro. An
intravenous injection of 0.1-1ng of TRH caused an elevation of blood TSH in mice within
2 minutes. The cold exposure stress and thyroidectomy increase blood TSH levels. Such
1
elevation of TSH is blocked by anti-TRH serum pretreatment, indicating the
involvement of TRH (1).
Estrogens: Estrogens are reported to enhance the TSH-releasing response of pituitary
cells for TRH-stimulation. In human subjects, women show larger TSH-releasing
response to TRH than men. The estrogen-pretreated men responded more to TRH
stimulation than control.
Estrogen is considered to increase the number of TRH
receptors in thyrotrophs (2).
Factors for suppression of secretion
Various factors have been reported to inhibit TSH secretion.
Somatostatin (Somatotropin or growth hormone release-inhibiting factor, SRIF):
Injection of anti-somatostatin causes an increase of TSH blood levels. Injection of
synthetic somatostatin to hypothyroidism patients lowers blood levels of TSH. In
healthy human subjects, somatostatin blocks the increase of TSH during sleeping.
Somatostatin suppressed the TSH-releasing action of TRH. These facts indicate the
direct action of somatostatin on TSH secretion in the pituitary. It was also reported that
somatostatin suppressed TRH action both in vivo and in vitro.
Also in rats,
somatostatin has reported to inhibit TSH surge in early morning (3).
Thyroid hormones (Triiodothyronin, T3, Thyroxin, T4): As an important factor, thyroid
hormones play the negative feedback role in hypothalamic-pituitary-thyroid system,
and also reported to directly suppress the action of TRH in the pituitary gland.
Growth hormone: Administration of GH lowers the reactivity of thyrotrophs to TRH.
Glucocorticoids: Cortisol injection suppresses TSH secretion in human. In human, the
blood levels of cortisol are low during 18:00~24:00 clock time, and TSH shows high
levels during this period, suggesting a relationship of circadian rhythm of TSH to that
of cortisol (4). This is supported by the fact that TSH releasing response to TRH at night
is higher than daytime (5). On the other hand, in rats, blood corticosterone levels are
high during dark period with lower TSH levels, and the lighting is started, TSH levels
go up, indicating suppressive effect of glucocorticoids on TSH secretion (6).
Melatonin: In rats, melatonin injection lowers blood TSH, T4 and T3 levels.
Opioid-peptides (Endorphins, Enkephalins), sympathetic stress, and starvation were
reported to decrease blood levels of TSH and thyroid hormones.
Structure and nature of TSH
TSH is a heterodimer glycoprotein hormone with molecular weight about 28,000, and
is consisted of α-subunit (96 amino acids in case of rat and mouse, with common
structure to LH and FSH) and β-subunit (118 amino acids, specific to TSH). Both
2
subunits are glycosylated (α-Subunit: at 56 and 82 asparagine, and β-subunit: at 23
asparagine).
Amino acid sequences of subunits of rat, mouse and dog TSH are shown below.
Boldface letters in the sequences indicate amino acid different from rat subunits.
Rat TSH α-subunit
lpdgdliiqg
cpecklkenk
yfsklgapiy
qcmgccfsra
yptparskkt
mlvpknitse
atccvaksft
katvmgnarv
enhtdchcst
cyyhks
↑Glycosylated
↑Glycosylated
Disulfide bonds: 11-35, 14-64, 32-86, 36-88, 63-91
Mouse TSH α-subunit
lpdgdfiiqg
cpecklkenk
yfsklgapiy
qcmgccfsra
yptparskkt
mlvpknitse
atccvakaft
katvmgnarv
enhtechcst
cyyhks
Homology: 94/96 (97.9%)
Dog TSH α-subunit
fpdgeftmqg cpecklkenk yfsklgapiy qcmgccfsra yptparskkt mlvpknitse
atccvakaft katvmgnakv enhtechcst cyyhks
Homology: 90/96 (93.8%)
Rat TSH β-subunit
fcipteymmy vdrrecaycl tintticagy cmtrdingkl flpkyalsqd vctyrdftyr
↑Glycosylated
tveipgcphh vapyfsypva lsckcgkcnt dysdctheav ktnyctkpqt fylggfsg
Disulfide bonds: 2-52, 16-67, 19-105, 27-83, 31-85, 88-95
Mouse TSH β-subunit
fcipteytmy vdrrecaycl tintticagy cmtrdingkl flpkyalsqd vctyrdfiyr
tveipgcphh vtpyfsfpva vsckcgkcnt dnsdciheav rtnyctkpqs fylggfsv
Homology 108/118 (91.5%)
Dog TSH β-subunit
fcfpteytmh verkecaycl tintticagy cmtrdingkl flpkyalsqd vctyrdfmyk
tveipgcprh vtpyfsypva vsckcgkcnt dysdciheai ktnyctkpqk syvvgfsi
Homology: 103/118 (87.3%)
Homology of amino acid sequences are very high between rat and mouse, and also dog
TSH shows a high homology to rat TSH.
Amino acid sequences of TSH of other species are shown in the last part of this text.
3
Nature of TSH
TSH shows microheterogeneity due to the differences in oligosaccharide chains, and
at least five components with different isoelectric points (9).
The author’s laboratory (Hormone Assay Center, Inst. of Endocrinology, Gunma Univ.,
in collaboration with the 1st Dept. of Int. Med., Sch. of Med., Gunma Univ., found an
interesting nature of TSH (9). After thyroidectomy of rats, the blood levels of
immunoreactive (IR) TSH rose up to 3 times after 2 days, 10 times after 1 week, and 20
times after 2 weeks compared with control levels (such a big increase is not seen with
LH after castration), and pituitary TSH contents decreased until 1 week, and then
recovered to nearly control levels after 2 weeks, and increased to 150% after 4 weeks.
Usually, β-subunit is present only in very small amount in the pituitary of normal rat.
But the amount of IR-β-subunit gradually increased after thyroidectomy, and exceeded
the amount of TSH after 4 weeks. A gel-filtration analysis of the pituitary homogenates
of thyroidectomized rats followed by β-subunit RIA showed a large peak in the fractions
with larger molecular weight than TSH, and this peak was low when assayed by TSH
RIA, indicating this big molecular weight component is not TSH but β-subunit-related
substance. If animals were given daily T4 injections after thyroidectomy at a dose
(1.5μg/100g body weight) that kept normal T4 levels, this β-subunit-related substance
did not appear. Such phenomenon was never seen with LH after castration, though LH
has similar structure to TSH.
Bioassay for TSH
A classical bioassay method by McKenzie (Endocrinology, 62; 372-382, 1958) was
based on the increase of radioiodine in the blood of immature mouse pretreated with
radioiodine and T4, while a recent in vitro method using cultured FRTL-5 cell, which is
derived from normal rat thyroid epithelial cell, with cAMP as a marker has been widely
used because of small variation (Sho, et al., Endocrinology; 124: 589-604, 1989, Pckles,
et al., J Mol Endocrinol, 9: 251-256, 1992)
Immunoassay for TSH
RIA and IRMA (immunoradiometric assay) kits have been commercially available for
human TSH. As for animal TSH, NIDDK’s assay kits for some animal species have been
supplied though they are not ready for use because users themselves must carry out
radioiodination.
An ELISA system for rat TSH is supplied by Shibayagi Co, Ltd., which will be
introduced in detail later.
4
Standard preparation and international unit of TSH
The amount of human TSH is expressed using international unit (IU). The first
international standard was 1st IRP human TSH (68/38), and defined as one ampoule
contained 150mIU. Then, 2nd IRP human TSH (80/558) was provided, one ampoule of
which contained 37mIU, and the weight of TSH was 7.5μg, and when compared with 1st
IRP. The assay value for TSH in clinical diagnosis is expressed as mIU/dl or μIU/ml
even immunoassay is used. The USP unit is almost equal to IU.
For animal TSH, a biological standard preparation, NIH-TSH-S1, of ovine origin, was
supplied by NIH. But as RIA has become more popular, immunoassay standards were
distributed and used widely. A rat TSH RIA kit has been provided by NIDDK belonging
to NIH. The kit consists of NIDDK rat TSH I-X, a highly purified preparation for
radioiodine labeling, NIDDK anti-rat TSH S-X, rabbit antiserum against rat TSH, and
NIDDK rat TSH RP-X, a standard preparation (X: lot number starting from 1). RP-1
was a very crude material, and the potency was only 1/160 of the preparation for
labeling. In such case, it was nonsense to express the assay values in terms of the
weight of RP-1 as some old reports did. After RP-1, RP-2 and RP-3 were provided, and
these preparations were prepared from I-series, i.e. highly purified for radioiodination,
by adding inert protective protein and buffer component, and if we reconstitute the
solution by adding purified water, we can get a standard solution of a fixed
concentration of the highly purified preparation. In this case we can use the weight of
RP-3 for assay results. The biological potency of NIDDK rat TSH I-9 (=RP-2 or 3) is
35IU/mg.
Some suggestive information for sampling
Circadian rhythm of blood TSH levels
Circadian rhythm is very well known with glucocorticoids and melatonin, but TSH
blood level shows also circadian rhythm, and considerable changes are seen during a
day. Changes in blood TSH levels will influence blood thyroid hormone levels. As a rat is
a nocturnal animal, blood TSH pattern is different from that of human TSH. The
representative glucocorticoid in human is cortisol, while in rat corticosterone is the only
glucocorticoid due to the absence of 17α-hydroxylase.
Presence of circadian rhythm means that the assay values will change depending on
the time of blood sampling.
In female rats, under the condition of 12 hours light-12 hours dark ( light on: 7:30,
off: 19:30 ), circadian changes were seen with TSH, T4 and T3. The peak of TSH was
5
seen immediately after the start of lighting, while peaks of T4 and T3 appeared 3-4
hours later, and corticosterone and prolactin increased soon after the light off. If the
light and dark periods are reversed, i.e. light off: 7:30 and light on: 19:30, for 3 weeks,
all the hormone peaks shifted 12 hours (6), suggesting that the circadian changes of
hypothalamic-pituitary-thyroid system are depending on the light and dark cycle.
According to this report, under the normal lighting condition, low blood level of TSH
during dark period started to increase after lighting to reach the maximum level about
2 hours later and kept the high level until 14:00, and then decreased until 16:00, and
the level remained low until next morning. T4 and T3 levels gradually increased after
onset of lighting, and reach the maximum at 16:00. So, the descending of TSH level
after 14:00 may be due to the negative feedback effect of thyroid hormones. But such
changes of TSH were not seen under reversed lighting condition. The relationship to
melatonin is also suggested.
Another report (8) described that, in 60 days old male rats, pituitary TSH content and
blood TSH level change in the opposite way, and the peak of the content and troughs of
blood TSH, T4, and T3 were seen at 24:00, while the trough of TSH content and peaks of
blood TSH, T4, and T3 were seen at 12:00.
From these data, it would be better to avoid sampling around the time of switching on
and off, and the sampling should be carried out at fixed period of time like 10:00 to
14:00 to stabilize control value. Circadian rhythm may change slightly depending on the
strain and sex of rats, I recommend checking the circadian change with your rats.
In human, blood TSH shows circadian rhythm, however, as human is not nocturnal
like rats, the situation is reverse. Cortisol shows the lowest level from 22:00, and start
increasing from 3:00, reaching the maximum level at 7:00-8:00. Blood TSH remains low
in the daytime, and starts ascending about 21:00 and remains high through the night.
Melatonin levels were low in the daytime, and rises from 23:00. In women, blood levels
of TSH were reported to increase from 21:00, reaching the peak at 24:00, then decrease
until 9:00, and stayed low until 21:00 (10).
Influence of stress and anesthesia on blood TSH level
I would like to introduce a report telling the effect of anesthesia on blood TSH which
appeared in Endocrinology, 1975
(13)
Anesthetics examined were Chloral hydrate 300mg/kg，i.p., Pentobarbitone
i.p., Thiopentone
Ether
inhalation
50mg/kg i.p., Urethane
(Deep
Methoxyflurane 1.5%
and
light
1.5g/kg (1/2 ip，1/2 sc)
anesthesia),
inhalation.
6
50mg/kg
Halothane
2%
inhalation,
Observations were made on simple administration, effects on TSH upraise by cold
exposure, and effects on TSH secretion by TRH administration.
After anesthetization, blood TSH levels were similar to those of controls, however
significantly decreased after 30 min.
Increase in TSH level by cold exposure was minimized by all anesthetics examined.
Especially ether, urethane and chloral hydrate completely blocked TSH increase, while
halothane and methoxyflurane retarded the increase.
TSH secretion by TRH was
inhibited only by deep ether
anesthesia, while thiopentone，
pentobarbitone
hydrate
and
chloral
enhanced
TRH
action.
Cold exposure causes TRH
release from hypothalamus,
and
thus
secreted
TRH
stimulate TSH release from
thyrotrophs
in
anterior
pituitary. The results mean
that all the anesthetics acted
on hypothalamus and block
TRH release. So, it is very
risky to use anesthetics in
experiments
which
include
hypothalamic function.
On
the
other
hand,
inhibition of TRH action was
seen
only
by
deep
ether
anesthesia, and some anesthetics seemed to enhance TRH action. In TRH experiments,
all the anesthetics including light ether anesthesia, except deep ether anesthesia,
showed no significant changes of TSH levels for the first 10 min after drug
administration. The deep ether anesthesia lowered TSH levels at 10 min after
anesthesia.
Another report (14) told the results as follows.
Non-anesthetized rats showed 5-fold increase in blood TSH level after 25 minutes’ cold
exposure, and ether anesthesia completely blocked this increase.
7
Pentobarbital anesthesia inhibited the effect of cold exposure by more than 90%.
Three minutes of ether anesthesia before cold exposure inhibited or retarded the
TSH-upraising reaction.
During 2 hours’ ether anesthesia at room temperature, blood TSH levels were
decreased.
Pentobarbital anesthesia at room temperature did not decrease blood TSH levels.
TRH administration under pentobarbital anesthesia increased TSH level more than
non-anesthetized control.
The response of blood TSH to TRH administration under ether anesthesia was less
than non-anesthetized control.
The slope of dose-response curve of blood TSH increase by TRH was larger under
pentobarbital anesthesia and smaller under ether anesthesia than that in
non-anesthetized control.
A report (15) told that immobilization stress for 10 minutes caused a decrease in blood
TSH, and further immobilization for 60 minutes the level went down to a half and the
level became 1/4 by 300 minutes’ immobilization.
Another report (16) showed that administration of apomorphine caused a decrease of
blood TSH level. Infusion with apomorphine (50μg/20μl/min.) lowered the TSH
releasing response by TRH. These results may suggest that apomorphine works directly
on pituitary.
Extraction of anterior pituitary hormones with high efficiency.
In estimation of the pituitary contents of hormones, their complete extraction from
the gland is indispensable. All the hormones are stored in secretory granules, and some
are hydrophobic. We examined various methods for the simple and complete extraction
of hormones (11).
We found that prolactin was the most difficult to extract, and that immunoreactivty of
GH was lost if high concentration of ethanol is present under neutral pH, and that
simple homogenization with neutral phosphate buffer is not enough even for
glycoprotein hormones.
As our conclusion, we found two methods: 1) to homogenize the gland with neutral
phosphate buffered saline (PBS) containing 1~2% urea followed by freezing and
thawing, and then centrifugation, and 2) to sonicate the gland with PBS containing
about 0.1% Triton X-100, followed by freezing and thawing, then centrifugation. The
centrifugation at 4,000rpm for 15 minutes will give a clear supernatant fluid. Dilution
of the pituitary extract differed depending on hormones and also sex. So, preliminary
8
test for the best dilution for assay is absolutely necessary.
Introduction to Rat TSH ELISA KIT (R-type) (AKRTS-010R)
Shibayagi is providing an ELISA (Enzyme Linked ImmunoSorbent Assay) kit for
measurement of rat TSH (thyroid-stimulating hormone) with high sensitivity using
Sandwich assay principle.
Features
(1) Highly sensitive assay with the standard range of 0.288~36ng/ml.
(2) This kit is for TSH in rat serum, culture medium and tissue extract.
(3) Assay sample volume is 10μl in the standard procedure.
(4) Assay format is 96 wells.
(5) Standard preparation is rat origin.
(6) Components of the kit are provided ready to use or in concentrated form.
Antibodies used in this kit
A monoclonal antibody that recognizes TSH β-subunit is solidified on the wells and
used as capture antibody, and a monoclonal antibody that recognizes α-subunit is used
as detection antibody.
Assay principle
In Shibayagi’s Rat TSH ELISA Kit (R-type), biotin-conjugated anti-TSH and
standard or sample are incubated in monoclonal anti-TSH antibody-coated wells. After
15~18 hours’ incubation and washing, HRP (horse radish peroxidase)-conjugated avidin
is added, and incubated for 30 minutes. After washing, HRP-complex remaining in wells
are reacted with a chromogenic substrate (TMB) for 30 minutes, and reaction is stopped
by addition of acidic solution, and absorbance of yellow product is measured
spectrophotometrically at 450 nm(sub-wavelength is 620nm). The absorbance is nearly
proportional to TSH concentration. The standard curve is prepared by plotting
absorbance against standard TSH concentrations. TSH concentrations in unknown
samples are determined using this standard curve.
Assay range
The range of standard curve：0.288~36ng/ml
9
Reagents supplied
Components
State
Amount
Use after washing
96 wells/1 plate
Concentrated. Use after dilution
200 μl/1 vial
（A）Anti-TSH-coated plate
（B）Standard rat TSH (360ng/ml)
（C）Buffer solution
Ready for use.
60 ml/1 bottle
（D）Biotin-conjugated anti-TSH
Concentrated. Use after dilution.
50 μl/1 vial
（Ｅ）HRP-conjugated avidin
Concentrated. Use after dilution.
100 μl/1 vial
（Ｆ）Chromogenic substrate reagent (TMB)
Ready for use.
12 ml/1 bottle
（H）Reaction stopper（1M H2SO4）
Ready for use.
12 ml/1 bottle
Concentrated. Use after dilution.
100 ml/1 bottle
Plate cover
－
1 plate
Instruction Manual
－
1 copy
（Ｉ）Concentrated washing buffer (10x)
Preparation of assay samples
This kit is principally intended to measure TSH in rat serum and plasma. Tissue
extracts and incubation or culture media can be also assayed for TSH if confirmed
by assay validation tests.
For preparation of plasma, we recommend to use EDTA-2Na at final concentration of
1mg/ml.
Deep ether anesthesia at blood sampling may lower blood TSH level, we recommend
barbiturates as anesthesia, though they also influence hypothalamus to minimize TRH
release (Endocrinology 99: 875-880, 1975).
High hemolysis (more than 120mg/dl hemoglobin) may influence assay results.
If sample is turbid or contains insoluble materials, centrifuge and use clear
supernatant fluid.
Organic solvents may influence assay results.
Assay samples soon after preparation.
Sample dilution should be carried out with the buffer solution of the kit using small
test tubes before assay.
Mix well, and pipette 50 μl of diluted sample into a well. In
the standard assay procedure, the dilution rate is 5x. The minimum dilution rate is
2.5x. Undiluted serum and plasma are not suitable because their pH and high protein
content may influence assay results.
Frozen stored samples should be thawed just before assay and mixed well to make
them homogenous.
If the presence of any interfering substances is suspected, confirm dilution linearity
10
using more 2 different dilutions or more.
Stability and storage of samples
In immediate assay, samples can be kept in a refrigerator, and brought to room
temperature just before assay. If they have to be kept for a long period, tightly close the
container and store lower than –35oC. Avoid repeated freezing and thawing.
Summary of Assay Procedure
Day 1
Anti-TSH-coated plate
↓Washing 4 times*
Biotin-conjugated anti-TSH
50μl
↓Shaking**
Diluted sample or standard solution
50μl
↓Shaking**, Reaction at 2～8℃,15～18hours（Standing***）
Day 2
Dilution of HRP-conjugated avidin with buffer of room
temperature to 200x
↓Washing 4 times*
HRP-conjugated avidin
100μl
↓Shaking**, reaction at room temp. 30min (standing***)
↓Washing 4 times*
Chromogenic substrate reagent (TMB)
100μl
↓Shaking**, reaction at room temp. 20min (standing***)
Reaction stopper（１M H2SO4）※Careful
100μl
↓Shaking**
Measurement of absorbance（450nm, sub 620nm）
Room temp：20～25℃
*Washing buffer volume：300 μl/well
Plate washer pressure：5～25ml/min（depending on nozzle diameter）
Be careful not to try wells after removal of liquid.
**Guideline of shaking：800rpm-10sec.×3 times
Sub-wavelength(reference wavelength):600～650nm
***Put a plate cover on the plate while the reaction after shaking.
11
Standard curve and assay validation
An example of standard curve
.
Precision of assay
Intra-assay variation (3 samples, 5 replicates assay,) Mean CV is less than 5 %.
Reproducibility
Inter-assay variation (3 samples, 4 days, assayed in 4 replicates )
Mean CV is
less than 5 %
Spike Recovery test
Standard TSH was added in 3 concentrations to 2 serum samples and were assayed
in duplicates.
The recoveries were 98.8 ~103%
Dilution test
Two serum samples were serially diluted and assayed in triplicates.
The dilution curves showed excellent linearity with R2 of 0.9977 and 0.9998.
Reference assay data
Rat TSH assay data: Mean 3.43 ng/ml, SD 2.34ng/ml
Serum samples obtained between 14:00 to 16:00 clock hour from 8 adult CD(SD)
male rats.
Species specificity of the kit
This kit showed 100% cross-reactivity with highly purified canine TSH preparation,
BRC canine TSH (12). This indicated theoretically that canine TSH could be measured
with this kit. Mouse TSH amino acid sequence showed the homology of 97% with
α-subunit and 91% with β-subunit to those of rat TSH, and mouse pituitary homogenate
12
showed a parallel dilution curve to the rat TSH standard curve suggesting the
possibility that mouse TSH may be also measurable by this kit. Of course, careful and
enough examinations are necessary to apply this kit to canine or mouse TSH assay.
Hormone specificity of the kit
We examined hormone and species specificity of the monoclonal antibodies used in
this kit. The results are shown in the table below.
The capture antibody did not react with the family hormones, LH, FSH, or CG. So,
these hormones are washed off after the first reaction. The capture antibody
cross-reacted with mouse TSH and, to some extent, with human TSH.
Reactivity of the capture antibody examined by dot blot test.
Dot
sample
Rat TSH
Capture antibody
Detection antibody
+++
+++
-
++
+++
+++
-
++
++
++
Rat LH/FSH/CG
Mouse TSH
Mouse LH/FSH/CG
Human TSH
+++ ：Well reacted， ++ ：Reacted，+ ：Weakly reacted，－：Not reacted
Reactivity in ELISA system
Reactivity was examined using the kit by adding hormones to the system and checked
if they give any assay value.
Hormones were added in an amount of 100ng/ml.
Hormones examined
Reactivity
Rat TSH
100%
Rat LH
Less than detection limit
Rat FSH
Less than detection limit
Rat GH
Less than detection limit
85%(＊)
Human TSH
*： Preparaton used: Acris Antibodies GmbH/PA1199 （Recombinant human TSH）
Potency＞４ IU/mg
(This potency seems to be very low compared with highly purified rat TSH that
has the potency of 35IU/mg. But this is a recombinant TSH without
glycosylation. Deglycosylated glycoprotein hormone can bind the receptor, and
immunologically active, however, its action is weak.).
.
13
Amino acid sequence of TSH subunits with their homology to rat TSH
Bold letters shows different amino acids from those of rat TSH
α-Subunit
Bull
TSH
α-subunit
fpdgeftmqg cpecklkenk yfskpdapiy qcmgccfsra yptparskkt mlvpknitse
atccvakaft katvmgnvrv enhtechcst cyyh
Homology: 84/96 (87.5%)
Pig
TSH
α-subunit
fpdgeftmqg cpecklkenk yfsklgapiy qcmgccfsra yptparskkt mlvpknitse
atccvakaft katvmgnarv enhtechcst cyyhks
Homology: 90/96 (93.8%)
Cat
TSH
α-subunit
fpdgeftmqg cpecklkenk yfsklgapiy qcmgccfsra yptparskkt mlvpknitse
atccvakaft katvmgnakv enhtechcst cyhhki
Homology: 86/96 (89.6%)
Horse
TSH
α-subunit
fpdgefttqd cpecklrenk yffklgvpiy qckgccfsra yptparsrkt mlvpknitse
stccvakafi rvtvmgnikl enhtqcycst cyhhki
Homology: 77/96 (80.2%)
Human
TSH
α-subunit
apdvqdcpec tlqenpffsq pgapilqcmg ccfsrayptp lrskktmlvq knXXXXvtss
etccvaksyn rvtvmggfkv enhtachcst cyyhks
Homology: 33/96 (34.4%)
β-Subunit
Bull
TSH
β-subunit
fcipteymmh verkecaycl tinttvcagy cmtrdvngkl flpkyalsqd vctyrdfmyk
taeipgcprh vtpyfsypva isckcgkcnt dysdciheai ktnyctkpqk symvgfsi
Homology: 102/118 (86.4%)
Pig
TSH
β-subunit
fcipteymmh verkecaycl tintticagy cmtrdfngkl flpkyalsqd vctyrdfmyk
tveipgcphh vtpyfsypva isckcgkcnt dysdciheai ktnyctkpqk syvlefsi
Homology: 103/118 (87.3%)
Cat
TSH
β-subunit
fcfpteymmh verkecaycl tintticagy cmtrdingkl flpkyalsqd vctyrdflyk
tveipgcphh vtpyfsypva vsckcgkcnt dysdciheai ktndctkpqk sdvvgvXX
14
Homology: 18/118 (84.7%)
Horse
TSH
β-subunit
fcipteymmh verkecaycl tintticagy cmtrdingkl flpkyalsqd vctyrdfmyk
tveipgcpdh vtpyfsypva vsckcgkcnt dysdciheai kanyctkpqk syvvefsi
Homology: 102/118 (86.4%)
Human
TSH
β-subunit
fcipteytmh ierrecaycl tintticagy cmtrdingkl flpkyalsqd vctyrdfiyr
tveipgcplh vapyfsypva lsckcgkcnt dysdciheai ktnyctkpqk sylvgfsv
Homology: 106/118 (89.8%)
References
1. Mori, M., Kobayashi, I., and Wakabayashi, K.
Suppression of serum thyrotropin (TSH) concetrations following thyroidectomy and
cold exposure by passive immunization with antiserum to thyrotropin-releasing
hormone (TRH) in rats.
Metabolism 27:1485-1490, 1978
2. Woeber, K. A., and Braverman, L. E.
”Contemporary Endocrinology”, Ingbar, S. H.(Ed.)
Vol. 1, Plenum Press, N.W., 1979, pp.77-117.
3. Guillemin, R.
Recent Prog. Horm. Res., 28, 229-286, 1977
4. Wilber, J. F., Utiger, R. D.
The effect of glucocorticoids on thyrotropin secretion.
J. Clin. Invest., 48, 2096, 1969
5. Weeke, J.
Scand. J. Clin. Lab. Invest., 33, 17, 1974
6. Ottenweller, J. E., and Hedge, G. A.
Diurnal variations of plasma thyrotropin, thyroxine, and triiodothyronine in female rats
are phase shifted after inversion of the photoperiod
Endocrinology, 111: 509-514, 1982
7. Ozturk, G., Coskun, S., Erbas, D., and Hasanoglu, E.
The effect of melatonin on liver superoxide dismutase activity, serum nitrate and
thyroid hormone levels.
Jpn J. Physiol.;50:149-53, 2000
8. Ooka-Souda, S., Draves, D. J., Timiras, P. S.
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Diurnal rhythm of pituitary-thyroid axis in male rats and the effect of adrenalectomy.
Endocri. Res. Commun. 6:43-56, 1979
9. Mori, M., Oshima, K., Fukuda, H., Kobayashi, I. , and Wakabayashi K.
Changes in the multiple components of rat pituitary TSH and TSHβ subunit
following thyroidectomy.
Acta Endocrinol., 105: 49-56, 1984
10. Spontaneous diurnal TSH secretion is enhanced in proportion to circulating leptin
in obese premenopausal women.
Petra Kok, P., Ferdinand Roelfsema , F., Frölich, M., Meinders, A., E. , Pijl, H.
J. Clin. Endocr. Metab. August 9, 2005 as doi:10.1210/jc.2005-0003
11. Ishikawa J., Fuse, Y., and Wakabayashi, K.
Choice of extraction procedure for estimation of anterior pituitary hormone content.
Endocrinol. Japon. 34, 755-767, 1987
12. Chiba, K., Kobayashi, H., and Wakabayashi, K.
Isolation and partial characterization of LH, FSH and TSH from canine pituitary gand.
Endocrine J., 44: 205-218,1997
13. Mannisto, P. T., Saarinen, A., and Ranta, T.
Anesthetics and thyrotropin secretion in the rat
Endocrinology 99: 875-880, 1976
14. Ohtake M, Bray GA.
Effects of pentobarbital anesthesia on thyroid function in the rat.
Horm Metab Res. 9: 146-149, 1977
15. Immobilization stress and prolactin secretion in male rats.
Kawakami, M., Higuchi, T., Matsuura, M.
Neuroendocrinology 29: 262-269, 1079
16. Studies on the inhibitory effect of apomorphine and bromocryptine basal and TRH
induced level of TSH and PRL in hypothyroid rats under pentobarbiturate anesthesia.
Langer, P., Mess, B., Foldes, O., Ruzsas, C., Brozmanova, H., Straussove, K., and
Gschwendtova, K.
Exp. Clin. Endocrinol., 83: 269-274, 1984
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