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Instructions for use

TSH canine ELISAAR E-8500

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TSH canine ELISA
1. INTRODUCTION
1.1 INTENDED USE
The TSH canine ELISA is an enzyme immunoassay for the quantitative measurement of canine TSH(thyrotropin) in serum and EDTA-plasma.
1.2 SUMMARY AND EXPLANATION

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Thyroid stimulating hormone (TSH, thyrotropin) in dogs is similar in function to TSH found in other mammalian
species, including humans. It is a glycoprotein produced by the anterior pituitary gland. Through its action on
the thyroid gland, it plays a major role in maintaining normal circulating levels of the iodothyronines, T4 andT3. The production and secretion of TSH is controlled by negative feedback from circulating T4 and T3, and by
the hypothalamic hormone TRH (thyrotropin releasing hormone). The TSH molecule is composed of two
nonidentical subunits,  and , that are bound together in a noncovalent manner. Within a species, the TSH 
subunit is structurally identical to the  subunits of the related glycoprotein hormones (LH, FSH and chorionicgonadotropin). The  subunit of TSH and the  subunits of the related hormones are structurally hormonespecific, and account for their unique biological activities.
Hypothyroidism is considered to be a common endocrine disorder in dogs, whereas hyperthyroidism in thisspecies is nearly unknown. Dogs mostly suffer from primary hypothyroidism, involving impaired production of
the thyroid hormones, T4 and T3. In this condition, elevated TSH levels are expected. Secondary or tertiary
hypothyroidism, where thyroid hormone production is low as a consequence of hypothalamic or pituitarydisease, is believed to account for less than 5% of canine hypothyroidism cases. In the latter conditions,decreased levels of TSH would be expected. Usually, hypothyroidism in dogs is suspected on the basis of clinicalhistory and the presence of decreased levels of thyroid hormones. However, suppressed thyroid hormone levels
are nonspecific indicators of the disease, since they are often observed in nonthyroid illnesses. The evaluationof thyroid function and the diagnosis of hypothyroidism in dogs can be greatly improved through the use of thevalid assay for the determination of canine TSH.

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2. PRINCIPLE

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The test kit is a solid phase enzyme immunometric assay (ELISA) in the microplate format, designed for thequantitative measurement of canine TSH. The microplate is coated with an antibody specific for canine TSH.
Standards and samples are pipetted into the antibody coated microplate. Afterwards, a horseradish peroxidaselabeled antibody is added. During a two hour incubation sandwich complexes consisting of the two antibodies
and the canine TSH is formed. Non-reactive components are removed by a washing step.
A chromogenic substrate, TMB (3,3',5,5'-Tetra-Methyl-Benzidine), is added to all wells. During a 30 minutes
incubation, the substrate is converted to a colored end product (blue) by the bound enzyme. Enzyme reaction is
stopped by dispensing of hydrochloric acid as stop solution (change from blue to yellow). The color intensity is
direct proportional to the concentration of canine TSH present in the sample. The optical density of the color
solution is measured with a microplate reader at 450 nm.

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3. WARNINGS AND PRECAUTIONSThe kit is strictly intended for veterinary use only. Use by staff, who is specially informed and trained inmethods which are carried out by use of immunoassays.
2. All blood components and biological materials should be handled as potentially hazardous in use and for
disposal. Follow universal precautions when handling and disposing of infectious agents.
3. Before starting the assay, read the instructions completely and carefully. Use the valid version of the
package insert provided with the kit. Be sure that everything is understood.
4. The microplate contains snap-off strips. Unused wells must be stored at 2-8°C in the sealed foil pouch
and used in the frame provided.
5. Pipetting of samples and reagents must be done as quickly as possible and in the same sequence foreach step.
6. Use reservoirs only for single reagents. This especially applies to the substrate reservoirs. Using areservoir for dispensing a substrate solution that had previously been used for the conjugate solution
may result in colored solution. Do not pour reagents back into vials as reagent contamination may occur.
7. Mix the contents of the microplate wells thoroughly to ensure good test results. Do not reuse microwells.
8. Do not let wells dry during assay; add reagents immediately after completing the rinsing steps.
9. Allow the reagents to reach room temperature (18-25°C) before starting the test. Temperature will affectthe absorbance readings of the assay. However, values for the patient samples will not be affected.
10. Never pipet by mouth and avoid contact of reagents and specimens with skin and mucous membranes.
11. Do not smoke, eat, drink, or apply cosmetics in areas where specimens or kit reagents are handled.
12. Wear disposable gloves when handling specimens and reagents. Microbial contamination of reagents or
specimens may give false results.

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13. Handling should be done in accordance with the procedure defined by an appropriate national biohazard
safety guideline or regulation.
14. Do not use reagents beyond expiry date as shown on the kit labels.
15. All indicated volumes have to be performed according to the protocol. Optimal test results are only
obtained when using calibrated pipettes and microtiterplate readers.
16. Do not mix or use components from kits with different lot numbers. It is advised not to exchange wells ofdifferent plates even of the same lot. The kits may have been shipped or stored under different
conditions and the binding characteristics of the plates may vary slightly.
17. Avoid contact with Stop Solution. It may cause skin irritation and burns.
18. Chemicals and prepared or used reagents have to be treated as hazardous waste according to thenational biohazard safety guideline and regulation.
19. For information please refer to Safety Data Sheets. Safety Data Sheets for this product are available
upon request directly from the manufacturer.

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4. REAGENTS

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4.1 REAGENTS PROVIDED

Microtiter Plate - Ready to use
12x8 (break apart) strips with 96 wells; Wells coated with an anti-canine TSH antibody.

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AR E-8531
Content:

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Standards - lyophilized, reconstitution required*Standard

Concentration

Volume/Vial

AR E-8501

Standard A

0 ng/ml

1 ml

seSymbolStandard B

0.2 ng/ml

1 ml

AR E-8503

Standard C

0.46 ng/ml

1 ml

AR E-8504

Standard D

1.05 ng/ml

1 ml

AR E-8505

Standard E

2.2 ng/ml

1 ml

AR E-8506

Standard F

5.2 ng/ml

1 ml

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highly purified canine TSH in serum

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Content:

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AR E-8502

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Cat. no.

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Listed below are approximate concentrations, please refer to vial labels for exact concentrations:

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*For reconstitution see “Reagent preparation”.

Enzyme Conjugate - Ready to use

AR E-8540

contains a horseradish peroxidase-labeled polyclonal anti canine TSH antibody, in abuffered matrix.

Volume:1 x 11 ml, red

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Content:

TMB Substrate Solution - Ready to use

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AR E-0055

contains tetramethylbenzidine (TMB) and hydrogen peroxide in a buffered matrix.

Volume:1 x 22 mltheContent:on

Stop Solution - Ready to use

contains 2 N Hydrochloric Acid solution.

useContent:
Volume:ly

AR E-0080

1 x 7 ml

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Hazards
identification:

H290 May be corrosive to metals.
H314 Causes severe skin burns and eye damage.
H335 May cause respiratory irritation.
Wash Solution - 10X concentrated

AR E-0030
Volume:1 x 50 ml

see “Reagent Preparation”

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4.2 MATERIALS REQUIRED BUT NOT PROVIDED










Microplate reader capable for endpoint measurements at 450 nm
Vortex mixer
Microplate mixer operating more than 600 rpm
Distilled water
Graduated cylinders for 500 ml
Plastic containers for storage of the wash solution
Adjustable pipette for up to 1000 µl
Dispenser or repeatable pipet for 100 µl and 200 µl

4.3 REAGENT PREPARATION

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Standards:
Reconstitute lyophilized Standard A through F with 1.0 ml dist. water 30 minutes before use.

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Wash Solution:
Dilute with 450 ml dist. water to a final volume of 500 ml.
The diluted Wash Solution is stable for 12 weeks at room temperature.

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4.4 STORAGE CONDITIONS

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When stored at 2°C to 8°C unopened reagents will be stable until expiration date. Do not use reagents beyond
this date. Opened reagents must be stored at 2-8 °C. After first opening the reagents are stable for 30 days ifused and stored properly. Microtiter wells must be stored at 2-8 °C. Take care that the foil bag is sealed tightly.
Store Standards refrigerated, they will be stable at 2-8 °C for 7 days after reconstitution. For longer storage
aliquot and freeze at -20 °C.
Protect TMB-Substrate Solution from light.

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4.5 DISPOSAL OF THE KITS

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The disposal of the kit must be made according to the national regulations. Special information for this productis given in the Safety Data Sheet.
4.6 DAMAGED TEST KITS

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In case of any severe damage of the test kit or components, the manufacturer has to be informed written,
latest one week after receiving the kit. Severely damaged single components should not be used for a test run.
They have to be stored until a final solution has been found. After this, they should be disposed according tothe official regulations.

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5. SAMPLE COLLECTION AND PREPARATION

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For determination of canine TSH serum and EDTA-plasma are the preferred sample matrices. The procedure
calls for 100 µl sample per well. The samples may be stored refrigerated at 2 - 8 °C for one week, or up to
two months at –20 °C. To avoid repeated thawing and freezing the samples should be aliquoted.
Samples expected to contain canine TSH concentrations higher than the highest Standard F should be diluted in
the Canine TSH Standard A before assay. The additional dilution step has to be taken into account for the
calculation of the results.

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6. ASSAY PROCEDURE

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6.1 GENERAL REMARKS All reagents and samples must be allowed to come to room temperature (18-25°C) before use. All reagents
must be mixed without foaming.
 Once the test has been started, all steps should be completed without interruption.
 Use new disposal plastic pipette tips for each standard, control or sample in order to avoid crosscontamination.
 Absorbance is a function of the incubation time and temperature. Before starting the assay, it isrecommended that all reagents are ready, caps removed, all needed wells secured in holder, etc. This will
ensure equal elapsed time for each pipetting step without interruption.
 As a general rule the enzymatic reaction is linearly proportional to time and temperature.
 Respect the incubation times as stated in this instructions for use.
 Duplicate determination of standards, controls and samples is recommended in order to identify potential
pipetting errors.
 For internal quality control we suggest to use Canine Control. For more information please contact the
manufacturer.

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6.2 ASSAY PROCEDURE
1. Prepare a sufficient number of microplate wells to accommodate Standards (A through F) and patient
samples in duplicates.
Pipet 100 µl of each Standard, Control and sample into the wells prepared.

3.

Add 100 µl of Enzyme Conjugate to all well.

4.

Rotate for 2 hours on a plate mixer (600 – 900 rpm).

5.

Discard the content of the wells and wash 4 times with 300 µl buffered wash solution.
Remove as much wash solution as possible by beating the microplate carefully.

6.

Add 200 µl of TMB-Substrate Solution to all wells.

7.

Incubate without shaking for 30 minutes in the dark.

8.

Add 50 µl of Stop Solution to each well and mix carefully.

9.

Determine the absorbance of each well at 450±10 nm.

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It is recommended to read the wells within 15 minutes.

6.3 CALCULATION OF RESULTS

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3.

Calculate the average absorbance values for each set of standards, controls and samples.
Construct a standard curve by plotting the mean absorbance obtained from each standard against its
concentration with absorbance value on the vertical (Y) axis and concentration on the horizontal (X) axis.
Using the mean absorbance value for each sample determine the corresponding concentration from thestandard curve.
Automated method: The results in the IFU have been calculated automatically using a 4 PL (4 Parameter
Logistics) curve fit. 4 Parameter Logistics is the preferred calculation method. Other data reduction functions
may give slightly different results.
The concentration of the samples can be read directly from this standard curve. Samples with concentrations
higher than that of the highest standard have to be further diluted. For the calculation of the concentrations
this dilution factor has to be taken into account.

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1.
2.

6.3.1 EXAMPLE OF TYPICAL STANDARD CURVE

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A
B
C
D
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F

Concentration(ng/ml)
0
0.2
0.46
1.05
2.2
5.2

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Standard

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The figure below shows typical results for TSH canine test kits. These data are intended for illustration only andshould not be used to calculate results from another run.
OD
(450nm)
0.071
0.233
0.423
0.852
1.569
3.092

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7. EXPECTED NORMAL VALUES

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Blood was collected from 30 apparently healthy dogs. A mean canine TSH value of 0.216 ng/ml was found, with
an absolute range of 0.017 to 0.591 ng/ml.
Laboratories should consider reference range limits as guidelines only. Because of differences which may exist
between laboratories and locales with respect to population, laboratory technique and selection of referencegroups, it is important for each laboratory to establish by similar means the appropriateness of adopting thereference range suggested here.

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8. QUALITY CONTROLGood laboratory practice requires that controls are run with each standard curve. A statistically significant
number of controls should be assayed to establish mean values and acceptable ranges to assure proper
performance.
It is recommended to use control samples according to state and federal regulations. The use of control
samples is advised to assure the day-to-day validity of results. Use controls at both normal and pathologicallevels.
The controls and the corresponding results of the QC-Laboratory are stated in the QC certificate included in thekit. The values and ranges stated on the QC sheet always refer to the current kit lot and should be used for
direct comparison of the results.
It is also recommended to make use of national or international Quality Assessment programs in order toensure the accuracy of the results.
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Employ appropriate statistical methods for analysing control values and trends. If the results of the assay do
not fit to the established acceptable ranges of control materials patient results should be considered invalid.
In this case, please check the following technical areas: Pipetting and timing devices, microtiter plate reader,
expiration dates of reagents, storage and incubation conditions, aspiration and washing methods. Afterchecking the above mentioned items without finding any error contact your distributor or the manufacturer
directly.
9. PERFORMANCE CHARACTERISTICS
9.1 ANALYTICAL SENSITIVITY

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The analytical sensitivity of the TSH canine ELISA was calculated by adding two standard deviations from themean of twenty-two (22) replicate analyses of Standard A. The analytical sensitivity of the assay is
0.00002 ng/ml.

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9.2 REPRODUCIBILITY
9.2.1 INTRA-ASSAY

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Sample 3
2.57
0.28
11.0
18

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Sample 2
0.92
0.09
9.9
18

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Sample 1
0.57
0.04
7.0
18

Mean (ng/ml)
SD (ng/ml)
CV (%)
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The intra-assay variation was determined by 18 replicate measurements of 3 serum samples within one runusing the TSH canine ELISA. The intra-assay variability is shown below:

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9.2.2 INTER-ASSAYInst

The inter-assay variation was determined by duplicate measurements of 3 serum samples in 10 different runs
using the TSH canine ELISA. The inter-assay variability is shown below:heSample 1

Sample 3

0.97
0.08
8.5
10

3.15
0.15
4.8
10

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9.3 LINEARITY

Sample 2

0.53
0.05
10.2
10

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Mean (ng/ml)
SD (ng/ml)
CV (%)
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In dilution experiments sera with high antibody concentrations were diluted with the Standard A and assayed in
the TSH canine kit.

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Sample2

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Dilution
Factor
1:2
1:4
1:8
1:2
1:4
1:8
1:2
1:4
1:8

measuredConcentration

expected
Concentration

Recovery[ng/ml][ng/ml]

[%]

1.81
0.92
0.45
0.22
2.91
1.50
0.72
0.37
3.41
1.76
0.79
0.37

0.91
0.45
0.23
1.46
0.73
0.36
1.71
0.85
0.43

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100
96
103
99
103
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10 LIMITATIONS OF PROCEDURE
Reliable and reproducible results will be obtained when the assay procedure is performed with a complete
understanding of the package insert instruction and with adherence to GLP (Good Laboratory Practice). Any
improper handling of samples or modification of this test might influence the results.
10.1 DRUG INTERFERENCES
Until today no substances (drugs) are known to us, which have an influence to the measurement of TSH in asample.
11 LEGAL ASPECTS

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11.1 RELIABILITY OF RESULTS
The test must be performed exactly as per the manufacturer’s instructions for use. Moreover the user muststrictly adhere to the rules of GLP (Good Laboratory Practice) or other applicable national standards and/or
laws. This is especially relevant for the use of control reagents. It is important to always include a sufficientnumber of controls within the test procedure for validating the accuracy and precision of the test.
The test results are only valid if all controls meet the specified ranges and all other test parameters are also
within the given assay specifications. In case of any doubt or concern please contact the manufacturer.

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11.2 THERAPEUTIC CENSEQUENCES
Therapeutic consequences should never be based on laboratory results alone even if all test results are inagreement with the items as stated under point 11.1. Any laboratory result is only a part of the total clinicalpicture of a patient.
Only in cases where the laboratory results are in acceptable agreement with the overall clinical picture of thepatient therapeutic consequences should be derived. The test result itself should never be the sole determinant
for deriving any therapeutic consequences.

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11.3 LIABILITY
Any modification of the test kit and/or exchange or mixture of any components of different lots from one test
kit to another could negatively affect the intended results and validity of the overall test. Such modification
and/or exchanges invalidate any claim for replacement.
Claims submitted due to customer misinterpretation of laboratory results subject to point 11.2. are also invalid.
Regardless, in the event of any claim, the manufacturer’s liability is not to exceed the value of the test kit. Anydamage caused to the test kit during transportation is not subject to the liability of the manufacturer.

Ruschig, S., Kraft, W.
Bestimmung von caninem Thyroidea-stimulierendem Hormon (cTSH) im Blutserum des Hundes und seine
Reaktion im TRH-Stimulationstest.
Tierärztl Prax 1996; 24: 479-483.
Iversen, L., Hoier, R., Jensen, A.L. , Skydsgaard, M., Koch, J.
Evaluation of the analytical performance on an enzyme immunometric assay (EIA) designed to measureendogenous thyroid-stimulating hormone (TSH) in canine serum samples.
J. Vet. Med. A 45 (1998): 93-98.
Ramsey, I.K., Evans, H., Herritage, M.E.
Thyroid-stimulating hormone and total thyroxine concentrations in euthyroid, sick euthyroid andhypothyroid dogs.
Small Animal Practice 38 (1997): 540-545.
Cortese, L., Oliva, G., Verstegen, J., Ciaramella, P., Persechino, A.
Hyperprolactinaemia and galactorrhoea associated with primary hypothyroidism in a bitch.
Small Animal Practice 38 (1997): 572-575.

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12. REFERENCES

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13. SHORT INSTRUCTION(all sample sizes given in µl)
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D

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F

Sample

0

0.20

0.46

1.05

2.20

5.20

Solution

Pipet

Standard

100

100

100

100

100

100

-

Pipet

Sample

-

-

-

-

-

-

100

Pipet

EnzymeConjugate

100

100

100

100

100

100

100

Substrate
Solution

200

200

200

200

Stop
Solution

50

50

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Incubate for
2h at RT
on a shaker

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Steps

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In

ng/ml

A

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MP Wellpr

Decant

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200

200

200

50

50

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Pipet

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Wash 4x with
300 µL ofbuffered wash
solution

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Incubate for
30 min at RTin the dark

50

50

50

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Pipet

Storagetemperature

Manufacturer

Expiry date

Batch code

Consult instructions
for useContent

CautionCatalogue
numberPlease

Symbols:

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Read at = 450 nm

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Contains sufficient for<n> tests

For research use
only!

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