ab108682 Thyroid Stimulating Hormone TSH …...ab108682 Thyroid Stimulating Hormone (TSH) Human...

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ab108682

Thyroid Stimulating

Hormone (TSH) Human

ELISA kit

Instructions for Use

For the quantitative measurement of Human Thyroid Stimulating Hormone (TSH) in plasma and serum. This product is for research use only and is not intended for in vitro diagnostic use.

Thyroid Stimulating

Hormone (TSH) Human

Human plasma

product is for research use only and is not

ab108682 Thyroid Stimulating Hormone (TSH) Human ELISA kit

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Table of Contents

1. Introduction 3

2. Assay Summary 4

3. Kit Contents 5

4. Storage and Handling 6

5. Additional Materials Required 6

6. Preparation of Reagents 6

7. Preparation and Collection of Specimens 8

8. Assay Method 9

9. Data Analysis 12

10. Limitations 15

11. Specificity 16

12. Troubleshooting 17


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1. Introduction

ab108682 Thyroid Stimulating Hormone (TSH) Human ELISA kit is

intended for the quantitative determination of Thyroid Stimulating

Hormone (TSH) in human plasma (citrate) or serum.

Thyroid-stimulating hormone (TSH or thyrotropin) is a glycoprotein

hormone synthesized and secreted by the anterior lobe of the

pituitary gland, which regulates the endocrine function of the thyroid.

Structurally, the glycoprotein hormones are related heterodimers

comprised of a common alpha subunit and a hormone-specific beta

subunit, which are non-covalently bound to one another. The alpha

subunit of TSH is identical to that of human chorionic gonadotropin

(HCG), luteinising hormone (LH) and follicle-stimulating hormone

(FSH). The beta subunit is TSH-specific, and therefore determines

its function.


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2. Assay Summary

ab108682 is based on the principle of a solid phase enzyme-linked

immunosorbent assay. The assay system utilizes two monoclonal

antibodies specific for distinct antigenic determinants on the TSH

beta subunit.

The first antibody is immobilized on the surface of the microtiter wells. The second antibody is conjugated to horseradish peroxidase.

The test sample is allowed to react simultaneously with the two antibodies, resulting in the TSH molecules being sandwiched between the solid phase and the enzyme-linked antibodies. After an incubation step, the wells are washed with Washing Solution to remove all unbound material.

The immune complex is visualized by adding Tetramethylbenzidine (TMB) substrate which gives a blue reaction product. The intensity of this product is proportional to the concentration of TSH in the specimen.

Sulphuric acid is added to stop the reaction. This produces a yellow endpoint colour. Absorbance at 450 nm is read using an ELISA microwell plate reader. The color intensity is directly proportional to the concentration of TSH present in the test sample.


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3. Kit Contents

• Antibody-Coated Wells: 12 break-apart 8-well snap-off strips

coated with anti-TSH mouse monoclonal antibodies; in re-

sealable aluminium foil.

• Stop Solution: 1 bottle containing 15 ml sulphuric acid, 0.2 mol/l;

ready to use; red cap.

• Washing Solution (20x conc.): 1 bottle containing 50 ml of a 20-

fold concentrated buffer for washing the wells; pH 7.2 ± 0.2;

white cap. contains 0.1 % Bronidox L after dilution.

• Anti-TSH Conjugate: 1 bottle containing 15 ml of peroxidase

labelled monoclonal antibodies to TSH; coloured red; ready to

use; black cap. Contains 0.2 % Bronidox L.

• TMB Substrate Solution: 1 bottle containing 15 ml 3,3’,5,5’-

tetramethylbenzidine (TMB); ready to use; yellow cap.

• TSH Standard Set: colored yellow, ready to use; yellow cap (1

set, 1 ml/vial); contains 0, 0.2, 0.5, 2.5, 5.0, 10.0 and 20 µlU/ml.

Contains 0.1 % Kathon.

• 1 Strip holder.

• 1 Cover foil.


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4. Storage and Handling

The reagents are stable up to the expiry date stated on the label

when stored at 2 - 8° Keep microtiter plate in a sealed bag with

desiccant to minimize exposure to damp air.

5. Additional Materials Required

• ELISA microwell plate reader, equipped for the measurement of

absorbance at 450/620 nm

• Incubator 37 °C

• Manual or automatic equipment for rinsing wells

• Pipettes to deliver 50 and 100 µl

• Vortex tube mixer

• Deionised or (freshly) distilled water

• Timer

6. Preparation of Reagents

1. All reagents should be allowed to reach room temperature (20-

25°C) before use.

2. Coated Snap-off Strips: The ready to use break-apart snap-off

strips are coated with monoclonal antibodies to TSH. Store at 2


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- 8 °C. Immediately after removal of strips, the remaining strips

should be resealed in the aluminum foil along with the desiccant

supplied and stored at 2 - 8 °C; stability until the expiry date..

3. Anti-TSH Conjugate: The bottle contains 15 ml of a solution

with anti-TSH horseradish peroxidase, buffer, stabilizers,

preservatives and an inert red dye. The solution is ready to use.

Store at 2 - 8. After first opening stability until the expiry date

when stored at 2 - 8 °C.

4. Standards: The vials labelled with Standard A, B, C, D, E, F

and G contain a ready to use standard solution. The standards,

calibrated in accordance with the WHO 3rd

IS for hTSH

(81/565), have the following concentrations:

Standard A: 0.0 µIU/ml

Standard B: 0.2 µIU/ml

Standard C: 0.5 µIU/ml

Standard D: 2.5 µIU/ml

Standard E: 5.0 µIU/ml

Standard F: 10.0 µIU/ml

Standard G: 20.0 µIU/ml

The solutions have to be stored at 2 - 8 °C and contain 0.1 %

Kathon. After first opening stability until the expiry date when

stored at 2 - 8 °C.


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5. Washing Solution (20x conc.): The bottle contains 50 ml of a

concentrated buffer, detergents and preservatives. Dilute

Washing Solution 1 + 19; e. g. 10 ml Washing Solution + 190 ml

fresh and germ free redistilled water. The diluted buffer will

keep for 5 days if stored at room temperature. Crystals in the

solution disappear by warming up to 37 °C in a water bath. After

first opening stability until the expiry date when stored at 2 to

8 °C.

6. TMB Substrate Solution: The bottle contains 15 ml of a

tetramethylbenzidine/hydrogen peroxide system. The reagent is

ready to use and has to be stored at 2 - 8 °C, away from the

light. The solution should be colorless or could have a slight

blue tinge. If the substrate turns into blue, it may have become

contaminated and should be thrown away. After first opening

stability until the expiry date when stored at 2 - 8 °C.

7. Stop Solution: The bottle contains 15 ml 0.2 M sulphuric acid

solution. This ready to use solution has to be stored at 2 - 8 °C.

After first opening stability until the expiry date.

7. Preparation and Collection of Specimens

1. Use human serum or plasma (citrate) samples with this assay.

2. If the assay is performed within 5 days after sample collection,

the specimens should be kept at 2 - 8 °C; otherwise they should


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be aliquoted and stored deep-frozen (-20 - -70 °C). If samples

are stored frozen, mix thawed samples well before testing. Avoid

repeated freezing and thawing.

3. Heat inactivation of samples is not recommended.

8. Assay Method

Test Preparation:

Please read the test protocol carefully before performing the assay.

Result reliability depends on strict adherence to the test protocol as

described. If performing the test on ELISA automatic systems we

recommend increasing the volume of Washing Solution from 300 µl

to 350 µl to avoid washing effects. Prior to commencing the assay,

the distribution and identification plan for all specimens and

standards should be carefully established. Select the required

number of microtiter strips or wells and insert them into the holder.

Please allocate at least:

1 well (e. g. A1) for the substrate blank, 7 wells (e. g. B1, C1, etc.) for Standard A, B, C, D, E, F and G

• It is recommended to determine standards and samples in

duplicate.

• Perform all assay steps in the order given and without any

appreciable delays between the steps.


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• A clean, disposable tip should be used for dispensing each

standard and sample.

• Adjust the incubator to 37 ± 1 °C.

Assay Procedure:

1. Dispense 50 µl of each Standard (A, B, C, D, E, F and G) and

samples into their respective wells. Leave well A1 for the

substrate blank.

2. Dispense 100 µl of Conjugate into each well except for the blank

(e. g. A1).

3. Cover wells with the foil supplied in the kit.

4. Incubate for 1 hour ± 5 min at 37 ± 1 °C.

5. When incubation has been completed, remove the foil, aspirate

the content of the wells and wash each well five times with 300

µl of Washing Solution. Avoid overflows from the reaction wells.

The soak time between each wash cycle should be > 5 sec. At

the end carefully remove remaining fluid by tapping strips on

tissue paper prior to the next step!

Note: Washing is critical. Insufficient washing results in poor

precision and falsely elevated absorbance values.

6. Dispense 100 µl of TMB Substrate Solution into each well.

7. Incubate for exactly 15 min at room temperature (20 - 25 °C)

in the dark.


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8. Dispense 100 µl Stop Solution into all wells in the same order

and at the same rate as for the TMB Substrate Solution. Any

blue color developed during the incubation turns into yellow.

9. Measure the absorbance of the specimen at 450/620 nm within

30 min after addition of the Stop Solution.

Measurement:

Adjust the ELISA Microwell Plate Reader to zero using the

standard 0.

If - due to technical reasons - the ELISA reader cannot be adjusted

to zero using the substrate blank in well A1, subtract the absorbance

value of well A1 from all other absorbance values measured in order

to obtain reliable results!

Measure the absorbance of all wells at 450 nm and record the

absorbance values for each standard and sample in the distribution

and identification plan.

Dual wavelength reading using 620 nm as reference wavelength is

recommended.

Where applicable calculate the mean absorbance values of all

duplicates.


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9. Data Analysis

A. Assay Validation CriteriaI

In order for an assay to be considered valid, the following criteria

must be met:

Standard Absorbance

Substrate Blank <0.100

A <0.100

B > Standard A

C > Standard B

D > Standard C

E > Standard D

F > Standard E

G >1.000

B. Calculation of Results

In order to obtain quantitative results in µIU/ml plot the (mean)

absorbance values of the Standards A – G (y-axis) on graph paper in


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a system of coordinates against their corresponding concentrations

(x-axis) and draw a standard calibration curve.

Read results from this standard curve employing the (mean)

absorbance values of each patient specimen.

All suitable computer programs available can be used for automated

result reading and calculation.

C. Typical Calibration Curve

D. Interpretation of results

Normal value ranges for this ELISA assay should be established by

each laboratory. The following values should be considered as a

guideline: Normal TSH range 0.3 to 4.5 µIU/ml.


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E. Sensitivity

The analytical sensitivity - defined as the apparent

concentration of the analyte that can be distinguished from the

zero calibrator - is < 0.1 µIU/ml.

F. Precision

Intra-assay n Mean (E) CV (%)

Serum 1 24 0.519 6.7

Serum 2 24 0.207 5.4

Serum 3 24 0.064 10.5

Inter-assay n Mean (µIU/ml) CV (%)

Serum 1 12 5.42 5.2

Serum 2 12 2.45 4.0

Serum 3 12 1.40 4.0

G. Correlation


was compared with a reference electrochemiluminescence


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immunoassay. 47 biological specimens were used (the values

ranged from approx. 0 - approx. 11 µIU/ml). The linear regression

curve was calculated.

Y= 1.06 x -0.11

r2= 0.97

H. Interferences

Interferences with hemolytic, lipemic or icteric sera are not observed

up to a concentration of 10 mg/ml hemoglobin, 5 mg/ml triglycerides

and 0.5 mg/ml bilirubin.

I. Hook Effect

No hook effect was observed applying up to 5,000 µIU/ml Thyroid

Stimulating Hormone.

10. Limitations

• Bacterial contamination or repeated freeze-thaw cycles of the

specimen may affect the absorbance values.


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11. Specificity

The following hormones were tested for cross-reactivity at

concentrations up to the levels indicated below. No cross-reactivity

was observed for any of the components.

Hormone tested Concentration

[mIU/ml]

Produced intensity equivalent to TSH

[µIU/ml]

LH (WHO 2

nd IS 80/552)

70 175 350

- -

<0.1

hCG (WHO 5

th IS 07/364)

1,000 5,000

25,000

- -

<0.1

FSH (WHO IS 83/575)

10 20 50

- <0.5 <0.5


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12. Troubleshooting

Problem Cause Solution

Poor standard curve

Improper standard dilution Confirm dilutions made correctly

Standard improperly reconstituted (if applicable)

Briefly spin vial before opening; thoroughly resuspend powder (if applicable)

Standard degraded Store sample as recommended

Curve doesn't fit scale Try plotting using different scale

Low signal

Incubation time too short Try overnight incubation at 4 °C

Target present below detection limits of assay

Decrease dilution factor; concentrate samples

Precipitate can form in wells upon substrate addition when concentration of target is too high

Increase dilution factor of sample

Using incompatible sample type (e.g. serum vs. cell extract)

Detection may be reduced or absent in untested sample types

Sample prepared incorrectly

Ensure proper sample preparation/dilution

Large CV Bubbles in wells Ensure no bubbles present prior to reading plate


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All wells not washed equally/thoroughly

Check that all ports of plate washer are unobstructed/wash wells as recommended

Incomplete reagent mixing Ensure all reagents/master mixes are mixed thoroughly

Inconsistent pipetting Use calibrated pipettes and ensure accurate pipetting

Inconsistent sample preparation or storage

Ensure consistent sample preparation and optimal sample storage conditions (eg. minimize freeze/thaws cycles)

High background

Wells are insufficiently washed

Wash wells as per protocol recommendations

Contaminated wash buffer Make fresh wash buffer

Waiting too long to read plate after adding STOP solution

Read plate immediately after adding STOP solution

Low sensitivity

Improper storage of ELISA kit

Store all reagents as recommended. Please note all reagents may not have identical storage requirements.

Using incompatible sample type (e.g. Serum vs. cell extract)

Detection may be reduced or absent in untested sample types

For further technical questions please do not hesitate to

contact us by email ([email protected]) or phone (select

“contact us” on www.abcam.com for the phone number for

your region).


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ab108682 Thyroid Stimulating Hormone TSH …...ab108682 Thyroid Stimulating Hormone (TSH) Human...

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