differentiating of hyperthyroidism from euthyroid population and

extending the usefulness of TSH measurements. This method is

a second-generation assay, which provides the means for
discrimination in the hyperthyroid-euthyroid range.

B. TSH Tracer Reagent13 ml/vial - Icon E

One (1) vial containing enzyme labeled affinity purified
polyclonal goat antibody, biotinylated monoclonal mouse IgG
in buffer, dye, and preservative. Store at 2-8C.

In this method, TSH calibrator, patient specimen or control is first

added to a streptavidin coated well. Biotinylated monoclonal and
enzyme labeled antibodies (Abs) are added and the reactants
mixed. Reaction between the various TSH antibodies and native
TSH forms a sandwich complex that binds with the streptavidin
coated to the well.

C. Light Reaction Wells 96 wells - Icon

One 96-well white microplate coated with streptavidin and
packaged in an aluminum bag with a drying agent. Store at
2-8C.

After the completion of the required incubation period, the

antibody bound enzyme-thyrotropin conjugate is separated from
the unbound enzyme-thyrotropin conjugate by aspiration or
decantation. The activity of the enzyme present on the surface of
the well is quantitated by reaction with a suitable substrate to
produce light.

Thyrotropin (TSH)
Test System
Product Code: 375-300

The employment of several serum references of known

thyrotropin levels permits construction of a dose response curve
of activity and concentration. From comparison to the dose
response curve, an unknown specimen's activity can be
correlated with thyrotropin concentration.

3.0 PRINCIPLE
1.0 INTRODUCTION
Intended Use: The Quantitative Determination of Thyrotropin
Concentration in Human Serum by a Microplate)
Chemiluminescence Immunoassay (CLIA)

2.0 SUMMARY AND EXPLANATION OF THE TEST

Measurement of the serum concentration of thyrotropin (TSH), a
glycoprotein with a molecular weight of 28,000 Daltons and
secreted from the anterior pituitary, is generally regarded as the
most sensitive indicator available for the diagnosis of primary and
secondary (pituitary) hypothyroidism (1, 2). The structure of
human TSH is similar to that of the pituitary and placental
gonadotropins, consisting of an 89-amino acid -subunit which is
similar or identical between these hormones and a 115-amino
acid -subunit, which apparently confers hormonal specificity.
The production of the 2 subunits is separately regulated with
apparent excess production of the -subunit. The TSH molecule
has a linear structure consisting of the protein core with
carbohydrate side chains; the latter accounts for 16% of the
molecular weight.
Increase in serum concentrations of TSH, which is primarily
responsible for the synthesis and release of thyroid hormones, is
an early and sensitive indicator of decrease thyroid reserve and in
conjunction with decreased thyroxine (T4) concentrations is
diagnostic of primary hypothyroidism. The expected increase in
TSH concentrations demonstrates the classical negative
feedback system between the pituitary and thyroid glands. That
is, primary thyroid gland failure reduces secretion of the thyroid
hormones, which in turn stimulates the release of TSH from the
pituitary.
Additionally, TSH measurements are equally useful in
differentiating
secondary
and
tertiary
(hypothalamic)
hypothyroidism from the primary thyroid disease. TSH release
from the pituitary is regulated by thyrotropin releasing factor
(TRH), which is secreted by the hypothalamus, and by direct
action of T4 and triiodothyronine (T3), the thyroid hormones, at
the pituitary. Increase levels of T3 and T4 reduces the response
of the pituitary to the stimulatory effects of TRH. In secondary and
tertiary hypothyroidism, concentrations of T4 are usually low and
TSH levels are generally low or normal. Either pituitary TSH
deficiency (secondary hypothyroidism) or insufficiency of
stimulation of the pituitary by TRH (tertiary hypothyroidism)
causes this. The TRH stimulation test differentiates these
conditions. In secondary hypothyroidism, TSH response to TRH is
blunted while a normal or delayed response is obtained in tertiary
hypothyroidism.
Further, the advent of immunoenzymometric assays has provided
the laboratory with sufficient sensitivity to enable the

Immunoenzymometric assay (Type 3)

The essential reagents required for an immunoenzymometric
assay include high affinity and specificity antibodies (enzyme
conjugated and immobilized), with different and distinct epitope
recognition, in excess, and native antigen. In this procedure, the
immobilization takes place during the assay at the surface of a
microplate well through the interaction of streptavidin coated on
the well and exogenously added biotinylated monoclonal antiTSH antibody.
Upon mixing monoclonal biotinylated antibody, the enzymelabeled antibody and a serum containing the native antigen,
reaction results between the native antigen and the antibodies,
without competition or stearic hindrance, to form a soluble
sandwich complex. The interaction is illustrated by the following
equation:
Enz
Ab(p) + AgTSH +

Btn

ka
Ab(m)

Enz
Btn
Ab(p)-AgTSH- Ab(m)

k-a
Btn

Ab(m) = Biotinylated Monoclonal Ab (Excess Quantity)

AgTSH = Native Ag (Variable Quantity)
ENZ
Ab(p)=Enzyme labeled Polyclonal Ab (Excess Quantity)
ENZ
Btn
Ab(p)-AgTSH- Ab(m)= Antigen-Antibodies Sandwich Complex
k
= Rate Constant of Association
a
k = Rate Constant of Dissociation
-a

Simultaneously, the complex is deposited to the well through te

high affinity reaction of streptavidin and biotinylated antibody. This
interaction is illustrated below:
Enz
Btn
Ab(p)-AgTSH- Ab(m)+ StreptavidinC.W. immobilized complex
StreptavidinC.W. = Streptavidin immobilized on well
Immobilized complex = sandwich complex bound to the solid surface

After equilibrium is attained, the antibody-bound fraction is

separated from unbound antigen by decantation or aspiration.
The enzyme activity, determined by reaction with a substrate that
generates light, in the antibody-bound fraction is directly
proportional to the native antigen concentration. By utilizing
several different serum references of known antigen values, a
dose response curve can be generated from which the antigen
concentration of an unknown can be ascertained.

4.0 REAGENTS
Materials Provided:
A. Thyrotropin Calibrators -- 1.0 ml/vial - Icons A-G
Seven (7) vials of references for TSH Antigen at levels of 0(A),
0.5(B), 2.5(C), 5.0(D), 10(E), 20(F) and 40(G) IU/ml. Store at
2-8C. A preservative has been added.
Note: The calibrators, human serum based, were
calibrated using a reference preparation, which was
assayed against the WHO 2nd IRP 80/558.

D. Wash Solution Concentrate 20 ml - Icon

One (1) vial containing a surfactant in Buffered Saline. A
preservative has been added. Store at 2-8C.
A

E. Signal Reagent A 7.0ml/vial - Icon C

One (1) bottle containing luminol in buffer. Store at 2-8C.
F. Signal Reagent B 7.0ml/vial - Icon CB
One (1) bottle containing hydrogen peroxide (H2O2) in buffer.
Store at 2-8C.
G. Package Insert.
Note 1: Do not use reagents beyond the kit expiration date.
Note 2: Avoid extended exposure to heat and light. Opened
reagents are stable for sixty (60) days when stored at
2-8C. Kit and component stability are identified on the
label.
Note 3: Above reagents are for a single 96-well microplate.
4.1 Required But Not Provided:
1. Pipette(s) capable of delivering 50l and 100l volumes with a
precision of better than 1.5%.
2. Dispenser(s) for repetitive deliveries of 0.100ml and 0.350ml
volumes with a precision of better than 1.5% (optional).
3. Microplate washer or a squeeze bottle (optional).
4. Microplate luminometer.
7. Absorbent Paper for blotting the microplate wells.
8. Plastic wrap or microplate cover for incubation steps.
9. Vacuum aspirator (optional) for wash steps.
10. Timer.
11. Storage container for storage of wash buffer.
12. Distilled or deionized water.
13. Quality Control Materials.

5.0 PRECAUTIONS
For In Vitro Diagnostic Use
Not for Internal or External Use in Humans or Animals
All products that contain human serum have been found to be
non-reactive for Hepatitis B Surface antigen, HIV 1&2 and HCV
antibodies by FDA required tests. Since no known test can offer
complete assurance that infectious agents are absent, all human
serum products should be handled as potentially hazardous and
capable of transmitting disease. Good laboratory procedures for
handling blood products can be found in the Center for Disease
Control / National Institute of Health, "Biosafety in Microbiological
and Biomedical Laboratories," 2nd Edition, 1988, HHS.
Safe Disposal of kit components must be according to local
regulatory and statutory requirements.

6.0 SPECIMEN COLLECTION AND PREPARATION

The specimens shall be blood, serum in type, and the usual
precautions in the collection of venipuncture samples should be
observed. For accurate comparison to established normal values,
a fasting morning serum sample should be obtained. The blood
should be collected in a plain red-top venipuncture tube with or
without gel barrier. Allow the blood to clot. Centrifuge the
specimen to separate the serum from the cells.
Samples may be refrigerated at 2-8C for a maximum period of
five (5) days. If the specimen(s) can not be assayed within this
time, the sample(s) may be stored at temperatures of -20C for
up to 30 days. Avoid use of contaminated devices. Avoid
repetitive freezing and thawing. When assayed in duplicate,
0.100ml of the specimen is required.

7.0 QUALITY CONTROL

Each laboratory should assay controls at levels in the
hypothyroid, euthyroid and hyperthyroid range for monitoring
assay performance. These controls should be treated as
unknowns and values determined in every test procedure
performed. Quality control charts should be maintained to follow
the performance of the supplied reagents. Pertinent statistical
methods should be employed to ascertain trends. The individual
laboratory should set acceptable assay performance limits. Other
parameters that should be monitored include the 80, 50 and 20%
intercepts of the dose response curve for run-to-run
reproducibility. In addition, maximum light intensity should be
consistent with past experience. Significant deviation from
established performance can indicate unnoticed change in
experimental conditions or degradation of kit reagents. Fresh
reagents should be used to determine the reason for the
variations.

8.0 REAGENT PREPARATION

1. Wash Buffer
Dilute contents of Wash Concentrate to 1000ml with distilled
or deionized water in a suitable storage container. Store
diluted buffer at 2-30C for up to 60 days.
2. Working Signal Reagent Solution - Store at 2 - 8C.
Determine the amount of reagent needed and prepare by
mixing equal portions of Signal Reagent A and Signal Reagent
B in a clean container. For example, add 1 ml of A and 1ml
of B per two (2) eight well strips (A slight excess of solution is
made). Discard the unused portion if not used within 36
hours after mixing. If complete utilization of the reagents is
anticipated, within the above time constraint, pour the contents
of Signal Reagent B into Signal Reagent A and label
accordingly.
Note: Do not use reagents that are contaminated or have
bacteria growth.

9.0 TEST PROCEDURE

Before proceeding with the assay, bring all reagents, serum
references and controls to room temperature (20 - 27 C).
**Test Procedure should be performed by a skilled individual
or trained professional**
1. Format the microplates wells for each serum reference,
control and patient specimen to be assayed in duplicate.
Replace any unused microwell strips back into the
aluminum bag, seal and store at 2-8C.
2. Pipette 0.050 ml (50l) of the appropriate serum reference,
control or specimen into the assigned well.
3. Add 0.100 ml (100l) of the TSH Tracer Reagent to each well.
It is very important to dispense all reagents close to the
bottom of the coated well.
4. Swirl the microplate gently for 20-30 seconds to mix and
cover.
5. Incubate 45 minutes at room temperature.
6. Discard the contents of the microplate by decantation or
aspiration. If decanting, tap and blot the plate dry with
absorbent paper.
7. Add 350l of wash buffer (see Reagent Preparation Section),
decant (tap and blot) or aspirate. Repeat four (4) additional
times for a total of five (5) washes. An automatic or manual
plate washer can be used. Follow the manufacturers
instruction for proper usage. If a squeeze bottle is
employed, fill each well by depressing the container
(avoiding air bubbles) to dispense the wash. Decant the
wash and repeat four (4) additional times.
8. Add 0.100 ml (100l) of working signal reagent solution to all
wells (see Reagent Preparation Section). Always add
reagents in the same order to minimize reaction time
differences between wells.
DO NOT SHAKE THE PLATE AFTER SUBSTRATE ADDITION

9. Incubate for five (5) minutes at room temperature in the dark.

10. Read the RLUs (Relative Light Units) in each well in a
microplate luminometer for at least 0.2seconds per well. The
results can be read within 30 minutes of adding the signal
solution.

10.0 CALCULATION OF RESULTS

11.0 Q.C. PARAMETERS

13.0 EXPECTED RANGES OF VALUES

A dose response curve is used to ascertain the concentration of

TSH in unknown specimens.
1. Record the RLUs obtained from the printout of the
microplate reader as outlined in Example 1.
2. Plot the RLUs for each duplicate serum reference versus the
corresponding TSH concentration in IU/ml on linear graph
paper.
3. Draw the best-fit curve through the plotted points.
4. To determine the concentration of TSH for an unknown, locate
the average RLUs for each unknown on the vertical axis of
the graph, find the intersecting point on the curve, and read
the concentration (in IU/ml) from the horizontal axis of the
graph (the duplicates of the unknown may be averaged as
indicated). In the following example, the average RLUs
(25677) of the unknown intersects the calibration curve at
7.1IU/ml TSH concentration (See Figure 1).

In order for the assay results to be considered valid the

following criteria should be met:
1. The Dose Response Curve should be within established
parameters.
2. Four out of six quality control pools should be within
established ranges.

A study of euthyroid adult population was undertaken to

determine expected values for the TSH AccuLite CLIA method.
The number and determined range are given in Table 1. A
nonparametric method (95% Percentile Estimate) was used.
TABLE I
Expected Values for the TSH AccuLite CLIA
(in IU/ml)
Number
85
0.42
Low Normal Range
5.45
High Normal Range
70% Confidence Intervals for 2.5 Percentile
Low Range
0.30 0.55
5.05 6.02
High Range

EXAMPLE 1

Cal B
Cal C
Cal D
Cal E
Cal F
Cal G
Ctrl 1
Ctrl 2
Sample

Value
(IU/ml)

Cal A

100
105
1290
1350
7663
7600
17878
17645
36315
34147
61811
62331
99820
100180
907
902
21870
21468
26231
25124

Mean
RLUs
(B)

RLUs
(A)

Well
Number

Sample
I.D.

A1
B1
C1
D1
E1
F1
G1
H1
A2
B2
C2
D2
E2
F2
G2
H2
A3
B3
C3
D3

102

1325

0.5

7631

2.5

17761

5.0

35231

10.0

62071

20.0

100000

40.0

905

0.34

21669

6.00

25677

7.1

The data presented in Example 1 and Figure 1 is for illustration

only and should not be used in lieu of a dose response curve
prepared with each assay. In addition, the RLUs of the calibrators
have been normalized to 100,000 RLUs for the G calibrator
(greatest light output). This conversion minimizes differences
caused by efficiency of the various instruments that can be used
to measure light.

Figure 1
120000
100000

RLU

80000
60000
40000
Sample

20000
0
0

10

20
30
TSH Values in IU/ml

40

50

The MSDS and Risk Analysis Form for this product is available on
request from Monobind Inc.
12.1 Assay Performance
1. It is important that the time of reaction in each well is held
constant to achieve reproducible results.
2. Pipetting of samples should not extend beyond ten (10)
minutes to avoid assay drift.
3. Highly lipemic, hemolyzed or grossly contaminated
specimen(s) should not be used.
4. If more than one (1) plate is used, it is recommended to
repeat the dose response curve.
5. The addition of signal reagent initiates a kinetic reaction,
therefore the signal reagent(s) should be added in the same
sequence to eliminate any time-deviation during reaction.
6. Failure to remove adhering solution adequately in the
aspiration or decantation wash step(s) may result in poor
replication and spurious results.
7. Use components from the same lot. No intermixing of
reagents from different batches.
8. Accurate and precise pipetting, as well as following the exact
time and temperature requirements prescribed are essential.
Any deviation from Monobinds IFU may yield inaccurate
results.
9. All applicable national standards, regulations and laws,
including, but not limited to, good laboratory procedures,
must be strictly followed to ensure compliance and proper
device usage.
10. It is important to calibrate all the equipment e.g. Pipettes,
Readers, Washers and/or the automated instruments used
with this device, and to perform routine preventative
maintenance.
11. Risk Analysis- as required by CE Mark IVD Directive
98/79/EC - for this and other devices, made by Monobind,
can be requested via email from Monobind@monobind.com.
12.2 Interpretation
1. Measurements and interpretation of results must be
performed by a skilled individual or trained professional.
2. Laboratory results alone are only one aspect for determining
patient care and should not be the sole basis for therapy,
particularly if the results conflict with other determinants.
3. For valid test results, adequate controls and other
parameters must be within the listed ranges and assay
requirements.
4. If test kits are altered, such as by mixing parts of different
kits, which could produce false test results, or if results are
incorrectly interpreted, Monobind shall have no liability.
5. If computer controlled data reduction is used to interpret the
results of the test, it is imperative that the predicted values for
the calibrators fall within 10% of the assigned concentrations.
6. Serum TSH concentration is dependent upon a multiplicity of
factors: hypothalamus gland function, thyroid gland function,
and the responsiveness of pituitary to TRH.
Thus,
thyrotropin concentration alone is not sufficient to
assess clinical status.
7. Serum TSH values may be elevated by pharmacological
intervention.
Domperiodone,
amiodazon,
iodide,
phenobarbital, and phenytoin have been reported to increase
TSH levels.
8. A decrease in thyrotropin values has been reported with the
administration of propranolol, methimazol, dopamine and dthyroxine (4).
9. Genetic variations or degradation of intact TSH into subunits
may affect the biding characteristics of the antibodies and
influence the final result. Such samples normally exhibit
different results among various assay systems due to the
reactivity of the antibodies involved.
"NOT INTENDED FOR NEWBORN SCREENING"

Substance
Thyrotropin (hTSH)
Follitropin (hFSH)
Lutropin Hormone (hLH)
Chorionic
Gonadotropin(hCG)

Cross
Reactivity
1.0000
< 0.0001
< 0.0001
< 0.0001

Concentration
1000ng/ml
1000ng/ml
1000ng/ml

15.0 REFERENCES
It is important to keep in mind that expected values for normal
population is dependent upon a multiplicity of factors: the
specificity of the method, the population tested and the precision
of the method in the hands of the analyst. For these reasons
each laboratory should depend upon the range of expected
values established by the Manufacturer only until an in-house
range can be determined by the analysts using the method with a
population indigenous to the area in which the laboratory is
located.

14.0 PERFORMANCE CHARACTERISTICS

14.1 Precision
The within and between assay precision of the TSH AccuLite
CLIA method were determined by analyses on three different
levels of pool control sera. The number, mean value, standard
deviation and coefficient of variation for each of these control sera
are presented in Table 2 and Table 3.
TABLE 2
Within Assay Precision (Values in IU/ml)

X
C.V.%
0.26
0.03
11.9
5.15
0.27
5.3
32.00
2.15
6.7
TABLE 3
Between Assay Precision* (Values in IU/ml)

Sample
Level 1
Level 2
Level 3

N
20
20
20

Sample
N
X
C.V.%
20
0.35
0.05
13.9
Level 1
20
5.42
0.52
9.6
Level 2
20
37.18
2.14
5.8
Level 3
*As measured in ten experiments in duplicate over ten days.
14.2 Sensitivity
The sensitivity (detection limit) was ascertained by determining
the variability of the 0 IU/ml serum calibrator and using the 2
(95% certainty) statistic to calculate the minimum dose. It was
determined to be 0.0062 IU/ml.
14.3 Accuracy
The TSH AccuLite CLIA assay was compared with a reference
Elisa assay. Biological specimens from hypothyroid, euthyroid
and hyperthyroid populations were used (The values ranged from
0.01IU/ml 41IU/ml). The total number of such specimens was
181. The least square regression equation and the correlation
coefficient were computed for this method in comparison with the
reference method. The data obtained is displayed in Table 4.
TABLE 4
Least Square
Mean
Regression
Correlation
Method
(x)
Analysis
Coefficient
14.97
y = 1.15 + 0.956 (x)
0.973
This
Method
14.44
Reference
Only slight amounts of bias between the TSH AccuLite CLIA
method and the reference method are indicated by the closeness
of the mean values. The least square regression equation and
correlation coefficient indicates excellent method agreement.

1. Hopton M.R., & Harrap, J.J., Immunoradiometric assay of thyrotropin as a

first line thyroid function test in the routine laboratory, Clinical Chemistry
32, 691. (1986)
2. Caldwell, G. et. Al., A new strategy for thyroid function testing, Lancet I,
1117. (1985)
3. Young, D.S., Pestaner, L.C., and Gilberman, U., "Effects of Drugs on
Clinical Laboratory Tests." Clinical Chemistry 21, 3660. (1975)
4. Spencer, CA, et al., Interlaboratory/Intermethod differences in Functional
Sensitivity of Immunometric Assays of Thyrotropin (TSH) and Impact on
Reliability of Measurement of Subnormal Concentrations of TSH, Clinical
Chemistry 41, 367. (1995)
5. Braverman, LE.:Evaluation of thyroid status in patients with thyrotoxicosis.
Clin. Chem. 42, 174-178. (1996)
6. Braverman, LE., Utigen, RD., Eds.: Werner and Ingbars The Thyroid A
th
Fundamental and Clinical Text 7 Ed. Philadelphia. Lippinscott-Raven.
(1996)
7. Degroot, LJ, Larsen, PR., Hennemenn, G.: Eds.The Thyroid and its
th
Diseases. 6 Ed. New York. Churchill Livingstone. (1996)
8. Fisher, DA.:Physiological variations in thyroid hormones: Physiological and
Pathophysiological considerations. Clin. Chem. 42, 135-139. (1996)
9. Beck-Peccoz, P., Persani, L.: Variable biological activity of thyroid
stimulating hormone. Eur.J.Endo 131,331-340. (1994)
10. Becker, DV., Bigos, ST., Gaitan, E.: Optimal use of blood tests for
assessment of thyroid function. JAMA 269, 2736-2740. (1989)
11. Fisher DA, Klein AH: Thyroid development and disorders of thyroid function
in the newborn. NEJM. 304, 702-712. (1981)
nd
12. Burtis CA. Ashwood ER (Ed): Tietz Textbook of Clinical Chemistry. 2 . Ed.
WB Saunders Company. Philadelphia, p 2208. (1994)
13. Alseveir RN, Gotlin RW: Handbook of Endocrine Tests in Adults and
nd
Children. 2 . Ed. Yearbook Publication. Chicago pg 22-25. (1978)
14. Magner JA: Thyroid Stimulating Hormone; Biosynthesis, Cell Biology and
bioactivity. Endo. Review 11, 35. 385. (1990)

Rev: 4

Date: 060712
Cat #: 375-300

Size

Reagent (fill)

Note: Computer data reduction software designed for

chemiluminescence assays may also be used for the data
reduction. If such software is utilized, the validation of the
software should be ascertained.

12.0 RISK ANALYSIS

14.4 Specificity
The cross-reactivity of this method to selected substances was
evaluated by adding the interfering substance to a serum matrix
at various concentrations. The cross-reactivity was calculated by
deriving a ratio between dose of interfering substance to dose of
thyrotropin needed to produce the same light intensity.

DCO:0538

96(A)

192(B)

480(D)

960(E)

A)

1ml set

1ml set

2ml set

2ml set x2

B)

1 (13ml)

2 (13ml)

1(60ml)

2 (60ml)
10 plates

C)

1 plate

2 plates

5 plates

D)

1 (20ml)

1 (20ml)

1 (60ml)

2 (60ml)

E)

1 (7ml)

2 (7ml)

1 (30ml)

2 (30ml)

F)

1 (7ml)

2 (7ml)

1 (30ml)

2 (30ml)