Dog TSHB / TSH-Beta ELISA

Kit
(Sandwich ELISA)

User Manual
Catalog No. LS-F12871
It is important that you read this entire manual
carefully before starting your experiment.

This kit is for Research Use Only. Not for Diagnostic Use.
This kit is not approved for use in humans or for clinical diagnosis.

Assay Specifications......................................................... 1
Assay Principle................................................................. 2
Kit Components............................................................... 4
Kit Storage....................................................................... 4
Other Required Supplies.................................................. 4
Experimental Layout........................................................ 5
Sample Collection............................................................ 6
Sample Collection Notes.................................................. 8
Sample Preparation......................................................... 9
Standard Preparation.................................................... 10
Reagent Preparation......................................................11
Reagent Preparation Notes........................................... 12
Assay Procedure............................................................ 13
Assay Procedure Notes.................................................. 14
Assay Procedure Summary............................................ 16
Calculation of Results.....................................................17
Troubleshooting Guide.................................................. 18

A SSAY S P E C I F I C A T I O N S
Target:

TSHB / TSH-Beta

Synonyms:

TSHB / TSH-Beta, TSHB, thyroid stimulating

hormone, beta, Thyrotropin alfa, Thyrotropin beta
subunit, TSH Beta, TSH-B, Thyrotropin beta chain,
Thyrotropin subunit beta, TSH-BETA, CHNG4

Specificity:

This kit is for the detection of Dog TSHB / TSH-Beta.

No significant cross-reactivity or interference
between TSHB / TSH-Beta and analogs was
observed. This claim is limited by existing
techniques therefore cross-reactivity may exist with
untested analogs.

Sample Types:

This kit is recommended for use with Dog Cell

Culture Supernatants, Plasma, Serum, and Tissue
Homogenates. Use with other sample types is not
supported.

Detection:

Colorimetric - 450nm

Detection Range:

1.56100 milli IU/ml

Sensitivity:

Typically less than 1.56 milli IU/ml

Performance:

Intra-Assay CV (<10%); Inter-Assay CV (<12%)

Limitations:

This kit is for Research Use Only and is not intended

for diagnostic use. This kit is not approved for use
in humans or for clinical diagnosis.

ASSAY P RINCIPLE
This assay is based on the sandwich ELISA principle. Each well of the
supplied microtiter plate has been pre-coated with a target specific
capture antibody. Standards or samples are added to the wells and the
target antigen binds to the capture antibody. Unbound Standard or
sample is washed away. A biotin-conjugated detection antibody is then
added which binds to the captured antigen. Unbound detection
antibody is washed away. An Avidin-Horseradish Peroxidase (HRP)
conjugate is then added which binds to the biotin. Unbound Avidin-HRP
conjugate is washed away. A TMB substrate is then added which reacts
with the HRP enzyme resulting in color development. A sulfuric acid
stop solution is added to terminate color development reaction and
then the optical density (OD) of the well is measured at a wavelength of
450 nm 2 nm. The OD of an unknown sample can then be compared
to an OD standard curve generated using known antigen concentrations
in order to determine its antigen concentration.

K IT C O M P O N E N T S
Component

Quantity

Coated 96-well Strip Plate

Standard (Lyophilized)
Sample Diluent
Assay Diluent A
Assay Diluent B
Detection Reagent A
Detection Reagent B
Wash Buffer (25x)
TMB Substrate
Stop Solution
Adhesive Plate Sealers
Instruction Manual

1
2 vials
1 vial x 20 ml
1 vial x 10 ml
1 vial x 10 ml
1 vial x 120 l
1 vial x 120 l
1 vial x 30 ml
1 vial x 10 ml
1 vial x 10 ml
4
1

K IT S T O R A G E
Upon receipt the kit should be stored at 4C if intended for use within
24 hours. Otherwise the Assay Plate, Standard, Detection Reagent A,
and Detection Reagent B should be stored at -20C. Avoid repeated
freeze-thaw cycles. Store all other kit components at 4C. The
Substrate should never be frozen. Once individual reagents are opened
it is recommended that the kit be used within 1 month. Unused Strip
Plate wells should be stored at -20C in a sealed bag containing
desiccant in order to minimize exposure to moisture. Do not use the kit
beyond its expiration date.

O THER R E Q U I R E D S U P P L I E S

Microplate reader with 450nm wavelength filter

High-precision pipette and sterile pipette tips
Eppendorf tubes
37C incubator
Deionized or distilled water
Absorbent paper

EXPERIMENTAL LAYOUT
The following is an example of how to layout a study. A dilution series
of the positive control Standard should be run in duplicate or triplicate,
with the last well in each series being the negative control blank.
Samples should also be run in duplicate or triplicate. Unknown samples
should be run as a dilution series in order to identify the optimal
dilution that produces an OD reading within the OD range of the
positive control Standard dilution series.
Example 1: Standard Curve and dilution series of an unknown sample.
1

Standard Dilution 1

Standard Dilution 1

Standard Dilution 2

Standard Dilution 2

Standard Dilution 3

Standard Dilution 3

Standard Dilution 4

Standard Dilution 4

Standard Dilution 5

Standard Dilution 5

Standard Dilution 6

Standard Dilution 6

Standard Dilution 7

Standard Dilution 7

Negative Control

Negative Control

3
Sample
(1:1)
Sample
(1:10)
Sample
(1:100)
Sample
(1:1k)
Sample
(1:10k)
Sample
(1:100k)
Sample
(1:1,000k)
Sample
(1:10,000k)

4
Sample
(1:1)
Sample
(1:10)
Sample
(1:100)
Sample
(1:1k)
Sample
(1:10k)
Sample
(1:100k)
Sample
(1:1,000k)
Sample
(1:10,000k)

4
Sample E
Sample E
Sample F
Sample F
Sample G
Sample G
Sample H
Sample H

Example 2: Standard Curve and samples run in duplicate.

A
B
C
D
E
F
G
H

1
Standard Dilution 1
Standard Dilution 2
Standard Dilution 3
Standard Dilution 4
Standard Dilution 5
Standard Dilution 6
Standard Dilution 7
Negative Control

2
Standard Dilution 1
Standard Dilution 2
Standard Dilution 3
Standard Dilution 4
Standard Dilution 5
Standard Dilution 6
Standard Dilution 7
Negative Control

3
Sample A
Sample A
Sample B
Sample B
Sample C
Sample C
Sample D
Sample D

S AMPLE C O L L E C T I O N
This assay is recommended for use with Dog Cell Culture Supernatants,
Plasma, Serum, and Tissue Homogenates. Use with other sample types
is not supported. The sample collection protocols below have been
provided for your reference.
Breast Milk - Centrifuge samples for 20 minutes at 1000g to remove
particulates. Collect the supernatant for assaying.
Cell Lysates - Collect and pellet the cells by centrifugation and remove
the supernatant. Wash the cells 3 times with PBS* then resuspend in
PBS*. Lyse the cells by ultrasonication 4 times. Alternatively freeze the
cells to -20C and thaw to room temperature 3 times. Centrifuge at
1500g for 10 minutes at 2 - 8C to remove cellular debris. Collect the
supernatant for assaying.
Erythrocyte Lysates - Centrifuge whole blood for 20 minutes at 1000g
to pellet the cells and remove the supernatant. Wash the cells 3 times
with PBS* then resuspend in PBS*. Freeze (-20C)/thaw (room
temperature) the cells 3 times. Centrifuge at 5,000g for 10 minutes at
2-8C to remove cellular debris. Collect the supernatant for assaying.
Erythrocyte lysates must be diluted with Sample Diluent before running.
Plasma - Collect plasma using heparin or EDTA as an anticoagulant.
Centrifuge samples for 15 minutes at 1000g at 28C within 30 minutes
of collection. Collect the supernatant for assaying.
Platelet-Poor Plasma - Collect plasma using heparin or EDTA as an
anticoagulant. Centrifuge samples for 15 minutes at 1000g at 28C
within 30 minutes of collection. It is recommended that samples should
be centrifuged for 10 minutes at 10,000g for complete platelet
removal. Collect the supernatant for assaying.
Sperm and Seminal Plasma - Allow semen to liquefy at room
temperature or 37C. After liquefaction, centrifuge at 2,000g for 10-15
minutes. Collect seminal plasma supernatant for assaying. Wash the
precipitated protein 3 times with PBS* then resuspend in PBS*. Lyse the
cells by ultrasonication then centrifuge at 2,000g for 10-15 minutes.
Collect the supernatant for assaying.
Serum - Use a serum separator tube and allow samples to clot for 2
hours at room temperature or overnight at 4C before centrifugation for
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20 minutes at approximately 1000g. Collect the supernatant for

assaying.
Tissue Homogenates - Because preparation methods for tissue
homogenates vary depending upon tissue type, users should research
tissue specific conditions independently. The following is one example
only. Rinse tissues in PBS* to remove excess blood and weigh before
homogenization. Finely mince tissues and homogenize them in 5-10mL
of PBS*with a glass homogenizer on ice. Lyse the cells by
ultrasonication or freeze (-20C)/thaw (room temperature) 3 times.
Centrifuge homogenate at 5000g for 5 minutes. Collect the
supernatant for assaying.
Urine - Aseptically collect the first urine of the day (mid-stream), voided
directly into a sterile container. Centrifuge to remove particulate matter
and collect the supernatant for assaying.
Cell culture supernatants, cerebrospinal, follicular, and lung lavage
fluids, saliva, sweat, tears, and other biological fluids - Centrifuge
samples for 20 minutes at 1000g to remove particulates. Collect the
supernatant for assaying.
* 1xPBS (0.02mol/L pH7.0-7.2)

S AMPLE C O L L E C T I O N N O T E S
1. LSBio recommends that samples are used immediately upon
preparation. Alternatively, samples stored at 2-8C should be used
within 5 days. For long-term storage sample aliquots should be
prepared and stored at -20C if used within 1 month, or -80C if
used within 6 months. Long term storage can result in protein
degradation and denaturation, which may result in inaccurate
results.
2. Avoid repeated freeze/thaw cycles for all samples.
3. In the event that a sample type not listed above is intended to be
used with the kit, it is recommended that the customer conduct
validation experiments in order to be confident in the results.
4. Due to chemical interference, the use of tissue or cell extraction
samples prepared by chemical lysis buffers may result in inaccurate
results.
5. Due to factors including cell viability, cell number, or sampling time,
samples from cell culture supernatant may not be detected by the
kit.
6. Samples should be brought to room temperature (18-25C) before
performing the assay without the use of extra heating.
7. Sample concentrations should be predicted before being used in the
assay. If the sample concentration is not within the range of the
standard curve, users must determine the optimal sample dilutions
for their particular experiments.
8. LSBio is responsible for the quality and performance of the kit
components but is NOT responsible for the performance of
customer supplied samples used with the kit.

S AMPLE P R E P A R A T I O N
The resulting Optical Density (OD) values of your sample must fall within
the OD values of the standard curve in order for the calculated antigen
concentration to be accurate. In many cases samples will need to be
diluted in order to lower the antigen concentration to sufficient levels.
Information about antigen concentrations within various sample types
may be available from the published literature; however, it is often
necessary to run a dilution series of each sample type. The following will
prepare sufficient volumes to run the Sample dilution series in triplicate.
In the case of small volume samples the first step in the series can be a
dilution, like 1:5 or 1:10, rather than undiluted sample. Running
duplicate or triplicate wells for each sample is recommended. * Always
dilute samples in the same buffer as the Standard used to generate
the Standard Curve.

S TANDARD P R E P A R A T I O N
The following are instructions for the preparation of a Standard dilution
series which will be used to generate the standard curve. The standard
curve is then used to determine the concentration of target antigen in
unknown samples (see the Calculation of Results section). The
following will prepare sufficient volumes to run the Standard dilution
series in duplicate. Reconstituted Standard and prepared standard
dilutions should be used immediately and not stored for future use.
Standard Stock Solution (100 milli IU/ml): Reconstitute 1 tube of
lyophilized Standard with 1.0 ml of Sample Diluent. Incubate at room
temperature for 10 minutes with gentle agitation (avoid foaming).
D1 (100 milli IU/ml):
Pipette 500l of Stock Standard into 0l of
Sample Diluent
D2 (50 milli IU/ml): Pipette 250l of D1 into 250l of Sample Diluent
D3 (25 milli IU/ml): Pipette 250l of D2 into 250l of Sample Diluent
D4 (12.5 milli IU/ml): Pipette 250l of D3 into 250l of Sample
Diluent
D5 (6.25 milli IU/ml): Pipette 250l of D4 into 250l of Sample
Diluent
D6 (3.125 milli IU/ml): Pipette 250l of D5 into 250l of Sample
Diluent
D7 (1.563 milli IU/ml): Pipette 250l of D6 into 250l of Sample
Diluent
Zero Standard (0 milli IU/ml): Use Sample Diluent alone

10

R EAGENT P R E P A R A T I O N
Bring all reagents to room temperature (18-25C) before use.
Detection Reagent A and B: Use the Detection Reagent A and B stocks
to prepare sufficient volumes of Detection Reagent A and B Working
Solution for the number of wells you are planning to run. Dilute
Detection Reagent A and B to a ratio of 1:100 using Assay Diluent A and
B respectively.
1x Wash Buffer: If crystals have formed in the concentrate, warm to
room temperature and mix it gently until crystals have completely
dissolved. Prepare 750 ml of Working Wash Buffer by diluting the
supplied 30 ml of 25x Wash Buffer Concentrate with 720 ml of
deionized or distilled water. Wash Buffer can be stored at 4C once
prepared.
TMB Substrate Solution: Using sterile techniques remove the needed
volume of TMB Substrate Solution for the number of wells you are
planning to run. Dispose of unused TMB Substrate Solution rather than
returning it to the stock container.

11

R EAGENT P REPARATION N O T E S
1. It is highly recommended that standard curves and samples are run
in duplicate within each experiment.
2. Once resuspended, standards should be used immediately, and
used only once. Long-term storage of reconstituted standards is
NOT recommended.
3. All solutions prepared from concentrates are intended for one-time
use. Do not reuse solutions.
4. Do not prepare Standard dilutions directly in wells.
5. Prepared Reagents may adhere to the tube wall or cap during
transport; centrifuge tubes briefly before opening.
6. All solutions should be gently mixed prior to use.
7. Reconstitute stock reagents in strict accordance with the
instructions provided.
8. To minimize imprecision caused by pipetting, ensure that pipettes
are calibrated. Pipetting volumes of less than 10L is not
recommended.
9. TMB Substrate solution is easily contaminated; sterility precautions
should be taken. TMB Substrate solution should also be protected
from light.
10. Do not substitute reagents from one kit lot to another. Use only
those reagents supplied within this kit.
11. Due to the antigen specificity of the antibodies used in this assay,
native or recombinant proteins from other manufacturers may not
be detected by this kit.

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A SSAY P R O C E D U R E
Bring all reagents and samples to room temperature without additional
heating and mix thoroughly by gently swirling before pipetting (avoid
foaming). Prepare all reagents, working standards, and samples as
directed in the previous sections.
1. Add 100l of Standard, Blank, or Sample per well, cover with a
plate sealer, and incubate for 2 hours at 37C.
2. Aspirate the liquid of each well, dont wash.
3. Add 100l of Detection Reagent A working solution to each well,
cover with a plate sealer, and gently agitate to ensure thorough
mixing. Incubate for 1 hour at 37C.
4. Aspirate the liquid from each well and wash 3 times. Wash by
adding approximately 350 l of 1x Wash Buffer using a squirt bottle,
multi-channel pipette, manifold dispenser or automated washer.
Allow each wash to sit for 1-2 minutes before completely aspirating.
After the last wash, aspirate to remove any remaining Wash Buffer
then invert the plate and tap against clean absorbent paper.
5. Add 100l of Detection Reagent B working solution to each well,
cover with a new plate sealer, and incubate for 60 minutes at 37C.
6. Aspirate the liquid from each well and wash 5 times as outlined in
step 4.
7. Add 90l of TMB Substrate solution to each well, cover with a new
plate sealer, and incubate for 15-30 minutes at 37C. Protect from
light and monitor periodically until optimal color development has
been achieved.
8. Add 50l of Stop Solution to each well. The blue color will change to
yellow immediately. If color change does not appear uniform, gently
tap the plate to ensure thorough mixing. The Stop Solution should
be added to wells in the same order and timing as the TMB
Substrate solution.
9. Determine the optical density (OD value) of each well immediately
using a microplate reader set to 450 nm.

13

A SSAY P R O C E D U R E N O T E S
1. ELISA Plate: Keep appropriate numbers of strips for 1 experiment
and remove extra strips from microtiter plate. Removed strips
should be placed in a sealed bag containing desiccant and stored at
-20C.
2. Solutions: In the event that Detection Reagent A working solution
appears cloudy, warm to room temperature and mix gently until
solution appears uniform. To avoid cross-contamination, change
pipette tips between additions of each standard, between sample
additions, and between reagent additions. Also, use separate
reservoirs for each reagent.
3. Applying Solutions: All solutions should be added to the bottom of
the ELISA plate well. Avoid touching the inside wall of the well.
Avoid foaming when possible.
4. Assay Timing: The interval between adding sample to the first and
last wells should be minimized. Delays will increase the incubation
time differential between wells, which will significantly affect the
experimental accuracy and repeatability. For each step in the
procedure, total dispensing time for addition of reagents or samples
should not exceed 10 minutes.
5. Incubation: To prevent evaporation and ensure accurate results,
proper adhesion of plate sealers during incubation steps is
necessary. Do not allow wells to sit uncovered for extended periods
of time between incubation steps. Do not let wells dry out at any
time during the assay. Strictly observe the recommended incubation
times and temperatures.
6. Washing: Proper washing procedure is critical. Insufficient washing
will result in poor precision and falsely elevated absorbance
readings. Residual liquid in the reaction wells should be patted dry
against absorbent paper during the washing process. Do not put
absorbent paper directly into the reaction wells.
7. Controlling Substrate Reaction Time: After the addition of the TMB
Substrate, periodically monitor the color development. Stop color
development before the color becomes too deep by adding Stop
Solution. Excessively strong color will result in inaccurate
absorbance reading.
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8. Reading: The microplate reader should be preheated and

programmed prior to use. Prior to taking OD readings, remove any
residual liquid or fingerprints from the underside of the plate and
confirm that there are no bubbles in the wells.
9. Reaction Time Control: Control reaction time should be strictly
followed as outlined.
10. Stop Solution: The Stop Solution contains an acid, therefore proper
precautions should be taken during its use, such as protection of the
eyes, hands, face, and clothing.
11. Mixing: During incubation times, the use of a micro-oscillator at low
frequency is recommended. Sufficient and gentle mixing is
particularly important in producing reliable results.
12. Kits from different batches may be a little different in detection
range, sensitivity, and color developing time. Please perform the
experiment exactly according to the supplied instructions.
13. Due to inter- and intra-assay variability, it is recommended that
appropriate carry-over controls be included between assays.
14. Prior to running valuable samples, LSBio recommends that the user
run a preliminary experiment using the supplied controls in order to
validate the assay.
15. To minimize external influence on the assay performance,
operational procedures and lab conditions (such as room
temperature, humidity, incubator temperature) should be strictly
controlled. It is also strongly suggested that the whole assay is
performed by the same operator from the beginning to the end.
16. The kit should not be used beyond the expiration date on the kit
label.

15

A SSAY P R O C E D U R E S U M M A R Y

Prepare all reagents, samples and standards.

Add 100 l of Sample, Standard, or Blank to each well and

incubate for 2 hours at 37C.
Aspirate and add 100 l of Detection Reagent A
and incubate of 1 hour at 37C.

Aspirate and wash 3 times.

Add 100 l of Detection Reagent B

and incubate for 60 minutes at 37C.

Aspirate and wash 5 times.

Add 90 l of TMB Substrate solution

and incubate for 15-30 minutes at 37C.

Add 50 l of Stop Solution.

Read immediately at 450nm.

16

C ALCULATION OF R E S U L T S
Average the duplicate readings for each standard, control, and sample
and subtract the average zero standard optical density. Create a
standard curve by reducing the data using computer software capable
of generating a four parameter logistic (4-PL) curve-fit. As an
alternative, construct a standard curve by plotting the mean absorbance
for each standard on the x-axis against the concentration on the y-axis
and draw a best fit curve through the points on the graph. The data may
be linearized by plotting the log of the target antigen concentrations
versus the log of the O.D. and the best fit line can be determined by
regression analysis. Use of a commercial software program such as
CurveExpert is recommended for performing these calculations. This
procedure will produce an adequate but less precise fit of the data. If
samples have been diluted, the concentration read from the standard
curve must be multiplied by the dilution factor.

Concentration (milli IU/ml)

Typical Data: The following standard curve is an example only and

should not be used to calculate results for tested samples. A new
standard curve must be generated for each set of samples tested.

Optical Density

17

TROUBLESHOOTING GUIDE
Problem

Possible Cause

Solution

Poor standard curve

Inaccurate pipetting

Check pipettes

Improper standard
dilution

Briefly spin the vial of

standard and dissolve
the powder
thoroughly by a gentle
mix.

Wells not completely

aspirated

Completely aspirate
wells between steps.

Too brief incubation

times

Ensure sufficient
incubation time.

Incorrect assay
temperature

Use recommended
incubation
temperature. Bring
substrate to room
temperature before
use.

Inadequate reagent
volumes

Check pipettes and

ensure correct
preparation.

Low signal

Improper dilution
Deep color but low
value

Plate reader settings

not optimal

Verify the wavelength

and filter setting in
the plate reader.
Open the Plate
Reader ahead to preheat.

18

Troubleshooting Guide (continued)

Problem

Possible Cause

Solution

Large CV

Inaccurate pipetting

Check pipettes.

High background

Concentration of
detector too high

Use recommended
dilution factor.

Plate is insufficiently
washed

Review the manual

for proper washing
instructions. If using a
plate washer, check
that all ports are
unobstructed.

Contaminated wash
buffer

Make fresh wash

buffer.

Improper storage of
the ELISA kit

All the reagents

should be stored
according to the
instructions.

Stop solution not

added

Stop solution should

be added to each well
before measurement.

Low sensitivity

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Important Note: During shipment, small volumes of product will occasionally become
entrapped in the seal of the product vial. We recommend briefly centrifuging the vial to
dislodge any liquid in the container's cap prior to opening.
Warning: This reagent may contain sodium azide and sulfuric acid. The chemical,
physical, and toxicological properties of these materials have not been thoroughly
investigated. Standard Laboratory Practices should be followed. Avoid skin and eye
contact, inhalation, and ingestion. Sodium azide forms hydrazoic acid under acidic
conditions and may react with lead or copper plumbing to form highly explosive metal
azides. On disposal, flush with large volumes of water to prevent accumulation.
Returns, Refunds, Cancelations: Any problems with LifeSpan products must be reported
to LifeSpan within 10 days of product receipt. The customer must obtain written
authorization from LifeSpan before returning items. To request that goods be returned,
please contact LifeSpan Technical Support. If an error by LifeSpan Biosciences results in
shipment of an incorrect order, LifeSpan will, at its option, either ship a replacement
order at no charge, or credit the customer's account for the original product shipped in
error. Returns and cancelations may be subject to a 30% restocking fee. Conditions &
Warranty: All LifeSpan products are intended for Research Use Only and are not for use
in human therapeutic or diagnostic applications. The information supplied with each
product is believed to be accurate, but no warranty or guarantee is offered for the
products, because the ultimate conditions of use are beyond LifeSpans control. The
information supplied with each product is not to be construed as a recommendation to
use this product in violation of any patent, and LifeSpan will not be held responsible for
any infringement or other violation that may occur with the use of its products. Under
no event will LifeSpan be responsible for any loss of profit or indirect consequential
damage, including, but not limited to, personal injuries resulting from use of these
products. LifeSpan's liability to any user of Products for damages that do not result from
any fault of the user, will be limited to replacement of the Product(s) only, and in no
event shall LifeSpan's liability exceed the actual price received by LifeSpan for the
Product(s) at issue. LifeSpan shall not be liable for any indirect, special, incidental or
consequential damages. LIFESPAN FURTHER DISCLAIMS ANY AND ALL EXPRESS AND
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BUT NOT LIMITED TO ANY IMPLIED WARRANTIES OF MERCHANTABILITY, FITNESS FOR A
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For Research Use Only. Not approved for use in humans or for clinical diagnosis.

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