MSD MULTI-SPOT Assay System
®
Muscle Injury Panel 3 (mouse) Kit
1-Plate Kit
K15186C-1
5-Plate Kit
K15186C-2
25-Plate Kit
K15186C-4
17865-v3-2014Jul | 1
MSD Toxicology Assays
Muscle Injury Panel 3 (mouse) Kit
cTnI, FABP3, Myl3, sTnI
This package insert must be read in its entirety before using this product.
FOR RESEARCH USE ONLY.
NOT FOR USE IN DIAGNOSTIC PROCEDURES.
MESO SCALE DISCOVERY®
A division of Meso Scale Diagnostics, LLC.
1601 Research Blvd.
Rockville, MD 20850 USA
www.mesoscale.com
MESO SCALE DISCOVERY, MESO SCALE DIAGNOSTICS, MSD, DISCOVERY WORKBENCH, MULTI-ARRAY, MULTI-SPOT, QUICKPLEX, SECTOR, SECTOR PR, SECTOR HTS, SULFOTAG, V-PLEX, S-PLEX, STREPTAVIDIN GOLD, MESO, www.mesoscale.com, SMALL SPOT (design), 96 WELL 1, 4, 7, & 10-SPOT (designs), 384 WELL 1 & 4-SPOT (designs), MSD
(design), V-PLEX (design), S-PLEX (design), and SPOT THE DIFFERENCE are trademarks and/or service marks of Meso Scale Diagnostics, LLC.
©2012-2014 Meso Scale Diagnostics, LLC. All rights reserved.
17865-v3-2014Jul | 2
Table of Contents
Introduction .......................................................................................................................................... 4
Principle of the Assay ........................................................................................................................... 5
Reagents Supplied ............................................................................................................................... 6
Required Material and Equipment (not supplied) ................................................................................ 6
Safety .................................................................................................................................................... 7
Reagent Preparation............................................................................................................................. 7
Protocol ................................................................................................................................................ 9
Validation and Verification .................................................................................................................. 10
Curve Fitting ....................................................................................................................................... 11
Typical Data ........................................................................................................................................ 12
Sensitivity............................................................................................................................................ 13
Precision ............................................................................................................................................. 13
Spike Recovery ................................................................................................................................... 14
Dilution Linearity ................................................................................................................................. 15
Specificity ........................................................................................................................................... 15
Stability ............................................................................................................................................... 15
Tested Samples .................................................................................................................................. 16
Assay Components ............................................................................................................................ 16
References .......................................................................................................................................... 17
Summary Protocol .............................................................................................................................. 19
Plate Diagrams ................................................................................................................................... 21
Ordering Information
MSD Customer Service
Phone:
Fax:
Email:
1-301-947-2085
1-301-990-2776
[email protected]
MSD Scientific Support
Phone:
Fax:
Email:
1-301-947-2025
1-240-632-2219 attn: Scientific Support
[email protected]
17865-v3-2014Jul | 3
Introduction
Troponin is a heterotrimer that regulates muscle contraction in skeletal and cardiac muscle but not in smooth muscle. Troponin
acts with intracellular calcium to control the interaction of actin and myosin filaments in striated muscle fibers. Though they perform
similar functions, cardiac and skeletal troponins differ in sequence and can be differentiated in immunoassays. Troponin I is an
inhibitory subunit that prevents muscle contraction in the absence of calcium. It is responsible for the binding of the troponin1
tropomyosin complex to actin. Troponin I exists in 3 isoforms which are found in slow-twitch (striated) skeletal muscle, fast-twitch
2
(striated) skeletal muscle, and cardiac muscle. Troponins are excellent biomarkers for myocardial injury in cardiotoxicity because
3
of their demonstrated tissue specificity.
Myosin light chain 3 (Myl3) is an essential light chain of the myosin molecule that is found in cardiac and slow-twitch skeletal
4
muscle. Myosin is a hexamer ATPase motor protein and a major constituent of thick muscle filament. It consists of a head domain
that “walks” along the actin chain to contract the muscle and a tail domain that is responsible for binding the myosin to its cargo.
Two heavy chain subunits intertwine to form the head and tail domains and 4 light chain subunits—2 regulatory light chains with
5
phosphorylation sites (encoded by the MYL2 genes) and 2 essential light chains (encoded by the MYL3 genes) —bind the heavy
chains together in the neck region between the head and tail domains. After damage to muscle tissue, myosin breaks down and
Myl3 becomes elevated in the blood. Myl3 can be used in conjunction with other toxicity biomarkers to confirm cardiac and slow
6
twitch skeletal muscle injury.
7
Fatty acid binding protein 3 (FABP3) is a monomeric protein that modulates the uptake of fatty acids in cells. Heart-type fatty
8
acid binding protein is released into circulation after myocardial ischemia and necrosis. FABP3 is mostly present in heart and
skeletal muscle but can also be found in brain, liver, and small intestine.
17865-v3-2014Jul | 4
Principle of the Assay
MSD toxicology assays provide a rapid and convenient method for measuring the levels of protein targets within a single, smallvolume sample. The assays in the Muscle Injury Panel 3 (mouse) Kit are sandwich immunoassays (Figure 1). MSD provides a plate
pre-coated with capture antibodies. The user adds the sample and a solution containing detection antibodies conjugated with
™
electrochemiluminescent labels (MSD SULFO-TAG ) over the course of one or more incubation periods. Analytes in the sample
bind to capture antibodies immobilized on the working electrode surface; recruitment of the detection antibodies by the bound
analytes completes the sandwich. The user adds an MSD buffer that provides the appropriate chemical environment for
electrochemiluminescence and loads the plate into an MSD instrument where a voltage applied to the plate electrodes causes the
captured labels to emit light. The instrument measures the intensity of emitted light to provide a quantitative measure of analytes in
the sample. This panel has been validated according to the principles outlined in “Fit-for-Purpose Method Development and
9
Validation for Successful Biomarker Measurement” by Lee, J.W. et al.
1.
2.
3.
4.
5.
6.
7.
cTnI
BSA Blocked
FABP3
BSA Blocked
Myl3
sTnI
BSA Blocked
Figure 1. Spot diagram showing placement of analyte capture antibodies. The numbering convention for the different spots is maintained in the
software visualization tools, on the plate packaging, and in the data files. A unique bar code label on each plate allows complete traceability back
to MSD manufacturing records.
17865-v3-2014Jul | 5
Reagents Supplied
Quantity per Kit
Product Description
Storage
K15186C-1
K15186C-2
K15186C-4
MULTI-SPOT 96-Well 7-Spot Muscle Injury Panel 3 (mouse) Plate
2–8°C
1 plate
5 plates
25 plates
SULFO-TAG Anti-ms cTnI Antibody
2–8°C
®
N75186A-1
1
(50X)
SULFO-TAG Anti-ms FABP3 Antibody
2–8°C
SULFO-TAG Anti-ms Myl3 Antibody
2–8°C
SULFO-TAG Anti-ms sTnI Antibody
2–8°C
Muscle Injury Panel 3 (mouse) Calibrator Blend
≤-70°C
Diluent 30
1
(50X)
1
(50X)
1
(50X)
(20X)
1 vial
(75 µL)
1 vial
(75 µL)
1 vial
(75 µL)
1 vial
(75 µL)
1 vial
1 vial
(375 µL)
1 vial
(375 µL)
1 vial
(375 µL)
1 vial
(375 µL)
5 vials
5 vials
(375 µL ea)
5 vials
(375 µL ea)
5 vials
(375 µL ea)
5 vials
(375 µL ea)
25 vials
(20 µL)
(20 µL ea)
≤-10°C
1 bottle
1 bottle
Diluent 33
≤-10°C
2 bottles
1 bottle
DTT
≤-10°C
1 vial
1 vial
(1 mL)
(1 mL ea)
EDTA pH 8.0
RT
1 bottle
1 bottle
5 bottles
Read Buffer T (4X)
RT
1 bottle
1 bottle
5 bottles
R50AB-4 (25 mL)
R50AD-4 (5 mL), R50AD-3 (50 mL)
(25 mM)
(0.5 M)
R92TC-3 (50 mL)
(25 mL)
(5 mL ea)
(1mL)
(4 mL)
(50 mL)
(25 mL)
(50 mL)
(4 mL)
(50 mL)
(20 µL ea)
5 bottles
(25 mL ea)
5 bottles
(50 mL ea)
5 vials
(4 mL ea)
(50 mL ea)
Required Material and Equipment (not supplied)
 Deionized water for diluting concentrated buffers
 Appropriately sized tubes for reagent preparation
 Microcentrifuge tubes for preparing serial dilutions
 Phosphate buffered saline plus 0.05% Tween-20 (PBS-T) for plate washing
 Liquid handling equipment for desired throughput, capable of dispensing 10 to 150 µL/well into a 96-well microtiter plate
 Plate washing equipment: automated plate washer or multichannel pipette
 Adhesive plate seals
 Microtiter plate shaker
1
SULFO-TAG–conjugated detection antibodies should be stored in the dark.
17865-v3-2014Jul | 6
Safety
Use safe laboratory practices and wear gloves, safety glasses, and lab coats when handling kit components. Handle and dispose of
all hazardous samples properly in accordance with local, state, and federal guidelines.
Reagent Preparation
Bring all reagents to room temperature. Thaw the stock calibrator blend on ice.
Important: Upon first thaw, separate Diluent 30 and Diluent 33 into aliquots appropriate for the size of your
needs before refreezing.
Prepare Diluent 33 + Additives
For the Muscle Injury Panel 3, samples and calibrators must be diluted in Diluent 33 to which EDTA and DTT have been added.
EDTA and DTT additive stocks are provided at the concentrations shown in the table below.
Additive
Stock Concentration
Final Concentration
EDTA
500 mM (16.7X)
30 mM (1X)
DTT
25 mM (100X)
0.25 mM (1X)
For 1 plate, combine:
 540 µL of EDTA stock solution
 90 µL of DTT stock solution
 8370 µL of Diluent 33
17865-v3-2014Jul | 7
Prepare Standards
MSD supplies a blended calibrator for the Muscle Injury Panel 3 (mouse) Kit at 20-fold higher concentration than the recommended
highest standard. We recommend a 7-point standard curve with 4-fold serial dilution steps and a zero calibrator blank. Signals from
the blank should be excluded when generating the curve. Thaw the stock calibrator and keep on ice, then add to diluent at room
temperature to make the standard curve solutions. To view the actual concentration of each calibrator in the blend, refer to the
certificate of analysis (COA) supplied with the kit. You may also find a copy of the lot-specific COA at www.mesoscale.com by
entering K15186C in the search box.
To prepare 7 standard solutions plus a zero calibrator blank for up to 4 replicates:
1) Prepare the highest standard by adding 15 µL of stock calibrator to 285 µL of Diluent 33 + additives. Mix well.
2) Prepare the next standard by transferring 60 µL of the highest standard to 180 µL of Diluent 33 + additives. Mix well.
Repeat 4-fold serial dilutions 5 additional times to generate 7 standards.
3) Use Diluent 33 + additives as the blank.
Dilute Samples
For mouse serum and plasma samples, MSD recommends a 4-fold dilution in Diluent 33 + additives; however, you may adjust
dilution factors for the sample set under investigation. Sample collection methods may affect the FABP3 endogenous levels.
To dilute sample 4-fold, add 25 µL of sample to 75 µL of Diluent 33 + additives.
Prepare Detection Antibody Solution
MSD provides each detection antibody in a 50X stock solution. The working detection antibody solution is 1X.
For 1 plate, combine:
 60 µL of 50X SULFO-TAG Anti-ms cTnI Antibody
 60 µL of 50X SULFO-TAG Anti-ms FABP3 Antibody
 60 µL of 50X SULFO-TAG Anti-ms Myl3 Antibody
 60 µL of 50X SULFO-TAG Anti-ms sTnI Antibody
 2760 µL of Diluent 30
Note: You may omit detection antibody for any analyte not being measured; add 60 µL of Diluent 30 for each omitted antibody.
Prepare Read Buffer
MSD provides Read Buffer T as a 4X stock solution. The working solution is 1X.
For 1 plate, combine:
 5 mL of Read Buffer T (4X)
 15 mL of deionized water
You may prepare diluted read buffer in advance and store it at room temperature in a tightly sealed container.
17865-v3-2014Jul | 8
Prepare MSD Plate
MSD plates are pre-coated with capture antibodies (Figure 1) and exposed to a proprietary stabilizing treatment to ensure the
integrity and stability of the immobilized antibodies. Plates can be used as delivered; no additional preparation (e.g., pre-wetting) is
required.
Protocol
1. Add Diluent 33 + Additives: Add 25 µL of Diluent 33 + additives to each
well. Seal the plate with an adhesive plate seal and incubate for 30 minutes
with vigorous shaking (300–1000 rpm) at room temperature.
Notes
Shaking the plate typically accelerates capture
at the working electrode.
2. Add Sample or Calibrator: Add 25 µL of calibrator or diluted sample per
well. Seal the plate with an adhesive plate seal and incubate for 2 hours
with vigorous shaking (300–1000 rpm) at room temperature.
You may prepare detection antibody solution during incubation.
3. Wash and Add Detection Antibody Solution: Wash the plate 3 times
with 300 µL/well of PBS-T. Add 25 µL of 1X detection antibody solution to
each well. Seal the plate with an adhesive plate seal and incubate for 2
hours with vigorous shaking (300–1000 rpm) at room temperature.
You may prepare diluted read buffer during incubation.
4. Wash and Read: Wash the plate 3 times with 300 µL/well of PBS-T. Add
150 µL of 1X Read Buffer T to each well. Analyze the plate on an MSD
instrument. No incubation in read buffer is required before reading the
plate.
You may keep excess diluted read buffer in a
tightly sealed container at room temperature
for later use.
Bubbles introduced when adding read buffer
will interfere with imaging of the plate and
produce unreliable data. Use reverse pipetting
technique to avoid creating bubbles.
Due to the varying nature of each research
application, you should assess assay stability
before allowing plates to sit with read buffer
for extended periods.
17865-v3-2014Jul | 9
Validation and Verification
MSD’s validation testing is conducted according to fit-for-purpose principles9 through a design-control process.
Validation. Bioanalytical and functional characterizations of calibrators, antibodies, and other assay components are completed to
ensure quality and consistency of reagents between lots. This includes plate coating uniformity and reagent and component
specificity testing for individual kit lots. Multiple control sample replicates in the specified matrices are tested to ensure the assay
meets MSD’s accuracy, precision, and sensitivity criteria.
Verification. Multi-day analysis with multiple runs per day using 6–12 plates is performed as part of the release testing for each lot.
 Curve Fitting
Calibration curve fitting methods, including weighting functions and 4- or 5-parameter logistic models, are evaluated on
multiple runs to select the best curve-fitting algorithm.
 Sensitivity and Dynamic Range
•
Sensitivity. The lower limit of detection (LLOD) is established based on runs throughout assay development. It
is a calculated concentration based on a signal 2.5 standard deviations above the average reading from the blank
calibrators. This results in a signal that is significantly higher than the background.
•
Dynamic Range. The dynamic (quantitative) range is established based on multiple runs from multiple lots. The
limits of the range—lower limit of quantification (LLOQ) and upper limit of quantification (ULOQ)— are the
lowest and highest concentrations that can be measured with acceptable levels of precision and accuracy.
The limits of quantification defined in this product insert are verified for each lot as part of the lot verification and quality
control release.
 Precision and Accuracy
Control samples made in the specified matrix are tested over multiple days to measure intra-run, inter-run, and inter-lot
accuracy and precision. Coefficient of variance (CV) information is presented in the product insert. During the validation
process, the assay is tested over multiple days with multiple runs per day using a total of 15–20 complete kits. Precision
and accuracy are verified for each lot as part of the lot verification and quality control release.
•
Precision. The typical specification for precision is a CV of less than 20% for controls on both intra- and interday runs.
•
Accuracy. The typical specification for accuracy includes a calculated concentration CV of less than 20%,
accuracy within 20% of expected concentration, and a total error of less than 30%.
 Robustness and Stability
Freeze-thaw testing and accelerated stability studies for calibrators, antibodies, and controls are performed during assay
development and are augmented with real-time stability studies on complete kits out to 24 months from the date of
manufacture.
17865-v3-2014Jul | 10
 Specificity, Spike Recovery, and Dilution Linearity
Assays are tested in the targeted matrix for non-specific bindings. Spike recovery and dilution linearity are tested across
the assay range to evaluate sample matrix effects.
 Tested samples
Normal samples for the specified species are tested to determine the normal range of biomarker concentration detected
with the assay.
Presented below are representative data from this kit’s validation process. The lot-specific standard curve and measured limits of
quantification can be found in the COA enclosed with each kit. You may also find a copy of the lot-specific COA at
www.mesoscale.com by entering your kit’s catalog number in the search box.
Curve Fitting
®
MSD DISCOVERY WORKBENCH software uses least-squares fitting algorithms to generate the standard curve that will be used to
calculate the concentration of analyte in the samples. The assays have a wide dynamic range (3–4 logs) that allows accurate
quantification without the need for dilution in many cases. By default, the software uses a 4-parameter logistic model (or sigmoidal
2
dose-response) and includes a 1/Y weighting function. The weighting function is important because it provides a better fit of data
over a wide dynamic range, particularly at the low end of the standard curve. The default algorithm provided optimal curve fitting for
this assay. Analysis using a 5-parameter logistic model does not improve goodness-of-fit.
17865-v3-2014Jul | 11
Typical Data
The following standard curves illustrate the dynamic range of the assay. Actual signals will vary. Best quantification of unknown
samples will be achieved by generating a standard curve for each plate using a minimum of 2 replicates of standards.
10000000
cTnI
FABP3
1000000
Myl3
Signal
sTnI
100000
Conc.
(ng/mL)
0
0.00630
0.0254
0.102
0.406
1.63
6.50
26.0
cTnI
Average
Signal
132
262
629
2225
8423
37 732
168 710
718 120
Conc.
(ng/mL)
0
0.0131
0.0525
0.210
0.841
3.36
13.5
53.8
Myl3
Average
Signal
160
223
305
598
1842
7978
33 296
138 068
10000
1000
100
0.001
0.01
0.1
1
10
100
Concentration (ng/mL)
1000
%CV
10.9
6.2
8.1
6.2
4.8
6.6
4.4
9.4
%CV
13.2
3.0
3.1
5.4
8.7
3.8
2.8
3.5
Conc.
(ng/mL)
0
0.0261
0.105
0.418
1.67
6.69
26.8
107
FABP3
Average
Signal
324
317
411
507
1494
9491
35 627
57 447
Conc.
(ng/mL)
0
0.0493
0.197
0.789
3.16
12.6
50.5
202
sTnI
Average
Signal
289
322
662
1902
6624
29 599
126 770
505 645
%CV
9.0
14.7
4.8
5.3
4.8
6.5
2.9
6.8
%CV
5.1
10.3
8.6
5.2
3.6
6.5
3.9
4.6
17865-v3-2014Jul | 12
Sensitivity
The lower limit of detection (LLOD) is a calculated concentration based on a signal 2.5 standard deviations above the background
(zero calibrator blank). The LLOD shown below was calculated based on 28 runs.
A multi-plate, multi-day study was performed to measure the reproducibility of the assay. The lower limit of quantification (LLOQ)
and upper limit of quantification (ULOQ) were established based on the results of multiple plate runs from multiple kit lots.
The LLOQ for FABP3, Myl3, and sTnI is the lowest concentration where the %CV of the calculated concentration is less than 20%
and the percent recovery is between 80% and 120% of the known value.
The LLOQ for cTnI is the lowest concentration where the %CV of the calculated concentration is less than 25% and the percent
recovery is between 75% and 125% of the known value.
The ULOQ for all the analytes is the highest concentration where the %CV of the calculated concentration is less than 20% and the
percent recovery of the standard is between 80% and 120% of the known value.
Average LLOD (ng/mL)
LLOQ (ng/mL)
ULOQ (ng/mL)
cTnI
FABP3
Myl3
sTnI
0.00880
0.0160
20.0
0.300
0.535
26.8
0.0479
0.135
41.4
0.0846
0.505
155
Precision
Controls were made by spiking calibrator into mouse serum at levels throughout the range of the assay. Analyte levels were
measured using a minimum of 3 replicates on 41 runs over 14 days.
Average intra-run %CV is the average %CV of the control replicates within an individual run.
Inter-run %CV is the variability of controls across 41 runs.
Inter-lot %CV is the variability of controls across 2 kit lots.
cTnI
FABP3
Myl3
sTnI
Control
Runs
Average
Conc.
(ng/mL)
Average
Intra-run
%CV
Inter-run
%CV
Inter-lot
%CV
High
Mid
Low
High
Mid
Low
High
Mid
Low
High
Mid
Low
41
41
41
41
41
41
41
41
41
41
41
41
11.0
1.58
0.430
12.6
3.25
1.25
26.9
6.98
0.489
49.9
8.25
1.34
6.1
4.7
4.2
6.8
6.0
6.2
5.1
5.3
5.2
6.3
4.6
4.9
8.4
8.6
9.3
10.9
9.3
12.9
8.0
7.1
10.0
10.2
10.0
11.0
4.8
5.6
6.7
10.0
7.9
12.5
3.7
1.8
7.9
4.7
4.0
7.9
17865-v3-2014Jul | 13
Spike Recovery
Normal mouse serum, EDTA plasma, and heparin plasma samples were diluted 4-fold and spiked with calibrators at multiple levels
throughout the range of the assay. The average percent recovery shown below was calculated from samples within the quantitative
range of the assay.
% Recovery=measured/expected∗100
cTnI
Sample
Type
Serum
(N=6)
EDTA
Plasma
(N=7)
Heparin
Plasma
(N=7)
FABP3
Spike
Conc.
(ng/mL)
Average
%Recovery
% Recovery
Range
Spike
Conc.
(ng/mL)
Average
%Recovery
% Recovery
Range
0.144–0.163
0.578–0.650
2.31–2.60
0.144–0.163
0.578–0.650
2.31–2.60
0.144–0.163
0.578–0.650
2.31–2.60
112
114
114
117
118
115
106
108
107
104–126
105–123
103–125
101–138
99–132
101–125
103–110
101–113
97–112
0.591–0.669
2.37–2.68
9.46–10.7
0.591–0.669
2.37–2.68
9.46–10.7
0.591–0.669
2.37–2.68
9.46–10.7
106
115
114
99
103
105
105
104
102
101–110
106–120
101–128
88–109
91–116
74–133
100–110
93–112
82–111
Myl3
Sample
Type
Serum
(N=6)
EDTA
Plasma
(N=7)
Heparin
Plasma
(N=7)
sTnI
Spike
Conc.
(ng/mL)
Average
%Recovery
% Recovery
Range
Spike
Conc.
(ng/mL)
Average
%Recovery
% Recovery
Range
0.336–0.346
1.35–1.38
5.38–5.53
0.336–0.346
1.35–1.38
5.38–5.53
0.336–0.346
1.35–1.38
5.38–5.53
121
136
140
129
135
132
129
142
138
104–137
127–157
126–163
113–140
112–157
124–140
124–142
132–150
121–154
1.08–1.26
4.33–5.05
17.3–20.2
1.08–1.26
4.33–5.05
17.3–20.2
1.08–1.26
4.33–5.05
17.3–20.2
103
102
97
101
99
92
103
97
89
90–110
91–109
89–102
88–110
86–107
81–101
92–113
85–106
76–96
17865-v3-2014Jul | 14
Dilution Linearity
To assess linearity, normal mouse serum, EDTA plasma, and heparin plasma samples were diluted 2-fold, 4-fold, 8-fold, and 16fold before testing. Percent recovery at each dilution was calculated by dividing the measured concentration by the expected
concentration, i.e., the concentration of the previous dilution. The average percent recovery shown below was calculated from
samples within the quantitative range of the assay.
% Recovery=measured/expected∗100
cTnI
Sample
Type
Fold
Dilution
Average
%Recovery
% Recovery
Range
FABP3
Average
%Recovery
% Recovery
Range
Myl3
Average
%Recovery
86
4
101
100–101
90
88–93
95
Serum
8
100
96–104
95
93–99
(N=3)
90
16
102
93–113
100
90–110
85
4
102
96–111
85
85*
EDTA
94
Plasma
8
103
101–107
91
91*
(N=3)
92
16
101
96–107
97
87–109
86
4
92
89–95
87
87*
Heparin
94
Plasma
8
94
92–96
93
91–94
(N=3)
-16
101
97–106
94
88–103
*A range of recovery cannot be provided since 2 of 3 samples were not within quantitative range.
-- Concentrations outside the quantitative range
% Recovery
Range
79–90
94–96
87–92
83–89
92–96
92*
85–87
89–99
--
sTnI
Average
%Recovery
92
100
-105
96
98
96
---
% Recovery
Range
90–94
100*
-104–107
96*
98*
94–98
---
Specificity
To assess specificity of the individual assays, the Muscle Injury Panel 3 (mouse) was run using blended antibodies with individual
calibrators (8.67 ng/mL cTnI; 35.7 ng/mL FABP3; 17.9 ng/mL Myl3; 67.3 ng/mL sTnI). No significant non-specific bindings were
observed.
Stability
Kit components were tested for freeze-thaw stability. Results (not shown) demonstrated that blended calibrator can be refrozen and
thawed 1 time without significantly affecting assay performance. Mouse serum and plasma samples can go through 5 freeze-thaw
cycles without any significant changes in their measured concentrations.
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Tested Samples
Serum, EDTA plasma, and heparin plasma samples were collected from normal CD-1 mice, diluted 4-fold, and tested with the
Muscle Injury Panel 3 (mouse). Median and range of concentrations for each sample set are displayed below. Concentrations are
corrected for sample dilution.
Sample
Type
Serum
EDTA
Plasma
Heparin
Plasma
Statistic
cTnI
FABP3
Myl3
sTnI
Median (ng/mL)
Range (ng/mL)
Number of Samples
Samples in Quantitative Range
Median (ng/mL)
Range (ng/mL)
Number of Samples
Samples in Quantitative Range
Median (ng/mL)
Range (ng/mL)
Number of Samples
Samples in Quantitative Range
0.771
<LLOQ–5.68
28
23
1.11
0.183–2.23
16
16
3.04
0.855–5.32
16
16
22.7
<LLOQ–>ULOQ
28
26
69.3
21.8–>ULOQ
16
11
98.1
42.3–>ULOQ
16
9
1.80
<LLOQ–24.6
28
21
1.83
0.652–20.3
16
16
2.04
0.792–5.08
16
16
<LLOQ
<LLOQ–11.1
28
8
<LLOQ
<LLOQ–21.5
16
3
<LLOQ
<LLOQ–4.06
16
5
Assay Components
Calibrators
In the Muscle Injury Panel 3 (mouse) calibrator blend, mouse cTnI, mouse FABP3, and mouse sTnI are native proteins. Full-length
recombinant Myl3 protein was expressed in E. coli.
Antibodies
Analyte
cTnI
FABP3
Myl3
sTnI
Source Species
MSD Capture Antibody
MSD Detection Antibody
Mouse Monoclonal
Mouse Monoclonal
Chicken Polyclonal
Mouse Monoclonal
Mouse Monoclonal
Mouse Monoclonal
Mouse Monoclonal
Mouse Monoclonal
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References
1.
Gomes AV, et al. The role of Troponin in muscle contraction. IUBMB Life. 2002 Dec;54(6):323-33.
2.
Marston SB, Redwood CS. Modulation of thin filament activation by breakdown or isoform switching of thin filament Proteins. Circ. Res. 2003 Dec
12;93(12):1170-8.
3.
Babuin L, Jaffe A S. Troponin: the biomarker of choice for the detection of cardiac injury. CMAJ. 2005 Nov 8;173(10):1191-02.
4.
Gao Y, et al. Myosin light chain kinase as a multifunctional regulatory protein of smooth muscle contraction. IUBMB Life, 2001 Jun;51(6):337-44.
5.
Kabaeva ZT, et al. Systematic analysis of the regulatory and essential myosin light chain genes: genetic variants and mutations in hypertrophic
cardiomyopathy. Eur J Hum Genet. 2002 Nov;10(11):741-8.
6.
Berna MJ, et al. Strategic use of immunoprecipitation and LC/MS/MS for trace-level protein quantification: myosin light chain 1, a biomarker of
cardiac necrosis. Anal Chem. 2007 Jun 1;79(11):4199-205.
7.
Kleine AH, et al. Release of heart fatty acid-binding protein into plasma after acute myocardial infarction in man. Mol. Cell. Biochem. 1992 Oct
21;116(1-2):155-62.
8.
Pritt ML, et al. Fabp3 as a biomarker of skeletal muscle toxicity in the rat: comparison with conventional biomarkers. Toxicol. Sci. 2008
Jun;103(2):382-96.
9.
Lee JW, et al. Fit-for-purpose method development and validation for successful biomarker measurement. Pharm Res. 2006 Feb;23(2):312-28.
17865-v3-2014Jul | 17
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Summary Protocol
MSD 96-well MULTI-SPOT Muscle Injury Panel 3 (mouse) Kit
MSD provides this summary protocol for your convenience.
Please read the entire detailed protocol prior to performing
the Muscle Injury Panel 3 (mouse) assays.
Sample and Reagent Preparation
 Bring all reagents to room temperature and thaw the calibrator on ice.
 Prepare Diluent 33 + additives.
 Prepare 7 standard solutions using the supplied calibrator:
• Dilute the stock calibrator blend 20-fold in Diluent 33 + additives.
• Perform a series of 4-fold dilution steps and prepare a zero calibrator blank.
 Dilute samples 4-fold in Diluent 33 + additives before adding to the plate.
 Prepare combined detection antibody solution by diluting each stock detection antibody 50-fold in Diluent 30.
 Prepare 1X Read Buffer T by diluting stock 4X Read Buffer T 4-fold with deionized water.
Step 1:
Add Diluent 33 + Additives
 Add 25 µL/well of Diluent 33 + additives.
 Incubate at room temperature with vigorous shaking (300–1000 rpm) for 30 minutes.
Step 2:
Add Sample or Calibrator
 Add 25 µL/well of calibrator of diluted sample.
 Incubate at room temperature with vigorous shaking (300–1000 rpm) for 2 hours.
Step 3:
Wash and Add Detection Antibody Solution
 Wash plate 3 times with 300 µL/well of PBS-T.
 Add 25 µL/well of 1X detection antibody solution.
 Incubate at room temperature with vigorous shaking (300–1000 rpm) for 2 hours.
Step 4:
Wash and Read Plate
 Wash plate 3 times with 300 µL/well of PBS-T.
 Add 150 µL/well of 1X Read Buffer T.
 Analyze plate on an MSD instrument.
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