Upload
others
View
3
Download
0
Embed Size (px)
Citation preview
www.abcam.com
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
1
ab108682 Thyroid Stimulating Hormone (TSH) Human ELISA kit
2
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
ab108682 Thyroid Stimulating Hormone (TSH) Human ELISA kit
3
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.
ab108682 Thyroid Stimulating Hormone (TSH) Human ELISA kit
4
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.
ab108682 Thyroid Stimulating Hormone (TSH) Human ELISA kit
5
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.
ab108682 Thyroid Stimulating Hormone (TSH) Human ELISA kit
6
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
ab108682 Thyroid Stimulating Hormone (TSH) Human ELISA kit
7
- 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.
ab108682 Thyroid Stimulating Hormone (TSH) Human ELISA kit
8
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
ab108682 Thyroid Stimulating Hormone (TSH) Human ELISA kit
9
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.
ab108682 Thyroid Stimulating Hormone (TSH) Human ELISA kit
10
• 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.
ab108682 Thyroid Stimulating Hormone (TSH) Human ELISA kit
11
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.
ab108682 Thyroid Stimulating Hormone (TSH) Human ELISA kit
12
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
ab108682 Thyroid Stimulating Hormone (TSH) Human ELISA kit
13
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.
ab108682 Thyroid Stimulating Hormone (TSH) Human ELISA kit
14
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
ab108682 Thyroid Stimulating Hormone (TSH) Human ELISA kit
was compared with a reference electrochemiluminescence
ab108682 Thyroid Stimulating Hormone (TSH) Human ELISA kit
15
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.
ab108682 Thyroid Stimulating Hormone (TSH) Human ELISA kit
16
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
ab108682 Thyroid Stimulating Hormone (TSH) Human ELISA kit
17
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
ab108682 Thyroid Stimulating Hormone (TSH) Human ELISA kit
18
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).
ab108682 Thyroid Stimulating Hormone (TSH) Human ELISA kit
19
Abcam in the USA Abcam in Japan
Abcam Inc Abcam KK
1 Kendall Square, Ste B2304 1-16-8 Nihonbashi
Cambridge, Kakigaracho,
MA 02139-1517 Chuo-ku, Tokyo
USA 103-0014
Japan
Toll free: 888-77-ABCAM (22226) Fax: 866-739-9884 Tel: +81-(0)3-6231-094 Fax: +81-(0)3-6231-0941
Abcam in Europe Abcam in Hong Kong
Abcam plc Abcam (Hong Kong) Ltd
330 Cambridge Science Park Unit 225A & 225B, 2/F
Cambridge Core Building 2
CB4 0FL 1 Science Park West Avenue
UK Hong Kong Science Park
Hong Kong
Tel: +44 (0)1223 696000
Fax: +44 (0)1223 771600 Tel: (852) 2603-682 Fax: (852) 3016-1888
Copyright © 2011 Abcam, All Rights Reserved. The Abcam logo is a registered trademark.
All information / detail is correct at time of going to print.