MSD MULTI-SPOT Assay System

Transcription

1 MSD MULTI-SPOT Assay System Muscle Injury Panel 3 (mouse) Kit 1-Plate Kit 5-Plate Kit 25-Plate Kit K15186C-1 K15186C-2 K15186C v3-2014Jul 1

2 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 Research Blvd. Rockville, MD USA MESO SCALE DISCOVERY, MESO SCALE DIAGNOSTICS, MSD, DISCOVERY WORKBENCH, MULTI-ARRAY, MULTI-SPOT, QUICKPLEX, SECTOR, SECTOR PR, SECTOR HTS, SULFO- TAG, V-PLEX, S-PLEX, STREPTAVIDIN GOLD, MESO, 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 Meso Scale Diagnostics, LLC. All rights reserved v3-2014Jul 2

3 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 Curve Fitting Typical Data Sensitivity Precision Spike Recovery Dilution Linearity Specificity Stability Tested Samples Assay Components References Summary Protocol Plate Diagrams Ordering Information MSD Customer Service Phone: Fax: CustomerService@mesoscale.com MSD Scientific Support Phone: Fax: attn: Scientific Support ScientificSupport@mesoscale.com v3-2014Jul 3

4 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 troponintropomyosin complex to actin. 1 Troponin I exists in 3 isoforms which are found in slow-twitch (striated) skeletal muscle, fast-twitch (striated) skeletal muscle, and cardiac muscle. 2 Troponins are excellent biomarkers for myocardial injury in cardiotoxicity because of their demonstrated tissue specificity. 3 Myosin light chain 3 (Myl3) is an essential light chain of the myosin molecule that is found in cardiac and slow-twitch skeletal muscle. 4 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 phosphorylation sites (encoded by the MYL2 genes) and 2 essential light chains (encoded by the MYL3 genes) 5 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 twitch skeletal muscle injury. 6 Fatty acid binding protein 3 (FABP3) is a monomeric protein that modulates the uptake of fatty acids in cells. 7 Heart-type fatty acid binding protein is released into circulation after myocardial ischemia and necrosis. 8 FABP3 is mostly present in heart and skeletal muscle but can also be found in brain, liver, and small intestine v3-2014Jul 4

5 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 Validation for Successful Biomarker Measurement by Lee, J.W. et al ctni 2. BSA Blocked 3. FABP3 4. BSA Blocked 5. Myl3 6. stni 7. 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 v3-2014Jul 5

6 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 N75186A-1 SULFO-TAG Anti-ms ctni Antibody 1 (50X) SULFO-TAG Anti-ms FABP3 Antibody 1 (50X) SULFO-TAG Anti-ms Myl3 Antibody 1 (50X) SULFO-TAG Anti-ms stni Antibody 1 (50X) Muscle Injury Panel 3 (mouse) Calibrator Blend (20X) Diluent 30 R50AB-4 (25 ml) Diluent 33 R50AD-4 (5 ml), R50AD-3 (50 ml) DTT (25 mm) EDTA ph 8.0 (0.5 M) Read Buffer T (4X) R92TC-3 (50 ml) 2 8 C 1 plate 5 plates 25 plates 2 8 C 2 8 C 2 8 C 2 8 C -70 C -10 C -10 C -10 C RT RT (75 µl) (75 µl) (75 µl) (75 µl) (20 µl) 1 bottle (25 ml) 2 bottles (5 ml ea) (1mL) 1 bottle (4 ml) 1 bottle (50 ml) (375 µl) (375 µl) (375 µl) (375 µl) 5 vials (20 µl ea) 1 bottle (25 ml) 1 bottle (50 ml) (1 ml) 1 bottle (4 ml) 1 bottle (50 ml) 5 vials (375 µl ea) 5 vials (375 µl ea) 5 vials (375 µl ea) 5 vials (375 µl ea) 25 vials (20 µl ea) 5 bottles (25 ml ea) 5 bottles (50 ml ea) 5 vials (1 ml ea) 5 bottles (4 ml ea) 5 bottles (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 v3-2014Jul 6

7 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 For 1 plate, combine: EDTA 500 mm (16.7X) 30 mm (1X) DTT 25 mm (100X) 0.25 mm (1X) 540 µl of EDTA stock solution 90 µl of DTT stock solution 8370 µl of Diluent v3-2014Jul 7

8 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 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 v3-2014Jul 8

9 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 Notes 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 ( rpm) at room temperature. 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 ( 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 ( 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 v3-2014Jul 9

10 Validation and Verification MSD s validation testing is conducted according to fit-for-purpose principles 9 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 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 v3-2014Jul 10

11 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 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 dose-response) and includes a 1/Y 2 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 v3-2014Jul 11

12 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. Signal ctni FABP3 Myl3 stni Concentration (ng/ml) ctni FABP3 Conc. Average Conc. Average %CV (ng/ml) Signal (ng/ml) Signal %CV Myl3 stni Conc. Average Conc. Average %CV (ng/ml) Signal (ng/ml) Signal %CV v3-2014Jul 12

13 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. ctni FABP3 Myl3 stni Average LLOD (ng/ml) LLOQ (ng/ml) ULOQ (ng/ml) 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 v3-2014Jul 13

14 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 Sample Type Serum (N=6) EDTA Plasma (N=7) Heparin Plasma (N=7) Spike Conc. (ng/ml) ctni Average %Recovery % Recovery Range Spike Conc. (ng/ml) FABP3 Average %Recovery % Recovery Range Sample Type Serum (N=6) EDTA Plasma (N=7) Heparin Plasma (N=7) Spike Conc. (ng/ml) Myl3 Average %Recovery % Recovery Range Spike Conc. (ng/ml) stni Average %Recovery % Recovery Range v3-2014Jul 14

15 Dilution Linearity To assess linearity, normal mouse serum, EDTA plasma, and heparin plasma samples were diluted 2-fold, 4-fold, 8-fold, and 16- fold 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 Sample Type Serum (N=3) EDTA Plasma (N=3) Heparin Plasma (N=3) Fold Dilution Average %Recovery ctni FABP3 Myl3 stni % Recovery Range Average %Recovery % Recovery Range Average %Recovery % Recovery Range Average %Recovery % Recovery Range * * * * * 98 98* * *A range of recovery cannot be provided since 2 of 3 samples were not within quantitative range. -- Concentrations outside the quantitative range 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 v3-2014Jul 15

16 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) <LLOQ Range (ng/ml) <LLOQ 5.68 <LLOQ >ULOQ <LLOQ 24.6 <LLOQ 11.1 Number of Samples Samples in Quantitative Range Median (ng/ml) <LLOQ Range (ng/ml) >ULOQ <LLOQ 21.5 Number of Samples Samples in Quantitative Range Median (ng/ml) <LLOQ Range (ng/ml) >ULOQ <LLOQ 4.06 Number of Samples Samples in Quantitative Range 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 Source Species Analyte MSD Capture Antibody MSD Detection Antibody ctni Mouse Monoclonal Mouse Monoclonal FABP3 Chicken Polyclonal Mouse Monoclonal Myl3 Mouse Monoclonal Mouse Monoclonal stni Mouse Monoclonal Mouse Monoclonal v3-2014Jul 16

17 References 1. Gomes AV, et al. The role of Troponin in muscle contraction. IUBMB Life Dec;54(6): Marston SB, Redwood CS. Modulation of thin filament activation by breakdown or isoform switching of thin filament Proteins. Circ. Res Dec 12;93(12): Babuin L, Jaffe A S. Troponin: the biomarker of choice for the detection of cardiac injury. CMAJ Nov 8;173(10): Gao Y, et al. Myosin light chain kinase as a multifunctional regulatory protein of smooth muscle contraction. IUBMB Life, 2001 Jun;51(6): 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 Nov;10(11): 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 Jun 1;79(11): Kleine AH, et al. Release of heart fatty acid-binding protein into plasma after acute myocardial infarction in man. Mol. Cell. Biochem Oct 21;116(1-2): Pritt ML, et al. Fabp3 as a biomarker of skeletal muscle toxicity in the rat: comparison with conventional biomarkers. Toxicol. Sci Jun;103(2): Lee JW, et al. Fit-for-purpose method development and validation for successful biomarker measurement. Pharm Res Feb;23(2): v3-2014Jul 17

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19 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: Step 2: Step 3: Step 4: Add Diluent 33 + Additives Add 25 µl/well of Diluent 33 + additives. Incubate at room temperature with vigorous shaking ( rpm) for 30 minutes. Add Sample or Calibrator Add 25 µl/well of calibrator of diluted sample. Incubate at room temperature with vigorous shaking ( rpm) for 2 hours. 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 ( rpm) for 2 hours. 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 v3-2014Jul 19

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