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Human TPO Kit Human TPO Kit 1-Plate Kit K151NWD-1 5-Plate Kit K151NWD-2 25-Plate Kit K151NWD-4 18043-v1-2013Aug | 1 MSD Biomarker Assays Human TPO Kit 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 Boulevard Rockville, MD 20850-3173 USA www.mesoscale.com MESO SCALE DISCOVERY, MESO SCALE DIAGNOSTICS, MSD, DISCOVERY WORKBENCH, MULTI-ARRAY, MULTI-SPOT, QUICKPLEX, SECTOR, SECTOR PR, SECTOR HTS, SULFO- TAG, V-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), and SPOT THE DIFFERENCE are trademarks and/or service marks of Meso Scale Diagnostics, LLC. ©2013 Meso Scale Diagnostics, LLC. All rights reserved. 18043-v1-2013Aug | 2 Table of Contents Introduction .................................................................................................................................................................. 4 Principle of the Assay ................................................................................................................................................... 4 Reagents Supplied ........................................................................................................................................................ 5 Required Material and Equipment (not supplied) ............................................................................................................ 5 Safety ........................................................................................................................................................................... 5 Reagent Preparation ...................................................................................................................................................... 6 Protocol ....................................................................................................................................................................... 8 Curve Fitting ................................................................................................................................................................. 8 Typical Data .................................................................................................................................................................. 9 Sensitivity .................................................................................................................................................................... 9 Assay Components ....................................................................................................................................................... 9 References .................................................................................................................................................................. 10 Summary Protocol ...................................................................................................................................................... 11 Plate Diagrams ........................................................................................................................................................... 13 Ordering Information MSD Customer Service Phone: 1-240-314-2795 Fax: 1-301-990-2776 Email: [email protected] MSD Scientific Support Phone: 1-240-314-2798 Fax: 1-240-632-2219 attn: Scientific Support Email: [email protected] 18043-v1-2013Aug | 3 Introduction Thrombopoietin (TPO), also known as megakaryocyte growth and development factor (MGDF), is a glycoprotein hormone that is the key regulator of megakaryopoiesis and thrombopoiesis.1 TPO is constitutively expressed by liver and bone marrow cells and is cleared from circulation by binding to the cMPL receptor.2 TPO stimulates bone marrow progenitor cells to proliferate and mature into megakaryocytes that produce platelets, which play critical roles in hemostasis.3 Circulating TPO levels may be useful in differentiating thrombocytopenias caused by peripheral destruction from those due to thrombopoietic failure: in the former, levels are depressed while in the latter levels are elevated.4 Elevated TPO levels may also serve as a biomarker for vascular damage and coronary angina. TPO may enhance platelet aggregation and platelet/monocyte interactions.5 Principle of the Assay MSD biomarker assays provide a rapid and convenient method for measuring the levels of protein targets within a single, small- volume sample. Human TPO is a sandwich immunoassay (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 a SECTOR® Imager 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. 1. TPO 2. BSA blocked 3. BSA blocked 4. 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 18043-v1-2013Aug | 4 Reagents Supplied Quantity per Kit Product Description Storage K151NWD-1 K151NWD-2 K151NWD-4 MULTI-SPOT 96-Well, 4-Spot Human TPO Plate 2–8°C 1 plate 5 plates 25 plates N451NWA-1 SULFO-TAG Anti-hu TPO Antibody1 2–8°C 1 vial 1 vial 5 vials (50X) (75 µL) (375 µL) (375 µL ea) Human TPO Calibrator ≤-70°C 1 vial 5 vials 25 vials (20X) (60 µL) (60 µL ea) (60 µL ea) Diluent 12 ≤-10°C 1 bottle 1 bottle 5 bottles R50JA-4 (10 mL), R50JA-3 (50 mL) (10 mL) (50 mL) (50 mL ea) Diluent 3 ≤-10°C 1 bottle 1 bottle 5 bottles R51BA-4(5 mL), R51BA-5 (25 mL) (5 mL) (25 mL) (25 mL ea) Blocker A Kit (Blocker A [dry] in 250 mL bottle and 50 mL bottle of 5X Phosphate Buffer) RT 1 kit 1 kit 5 kits R93AA-2 (250 mL) (250 mL) (250 mL) (250 mL ea) Read Buffer T (4X) RT 1 bottle 1 bottle 5 bottles R92TC-3 (50 mL) (50 mL) (50 mL) (50 mL ea) Required Material and Equipment (not supplied) 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 Deionized water 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. Additional safety information is available in the product Material Safety Data Sheet, which can be obtained from MSD Customer Service. 1 SULFO-TAG–conjugated detection antibodies should be stored in the dark. 18043-v1-2013Aug | 5 Reagent Preparation Bring all reagents to room temperature. Thaw the stock calibrator on ice. Important: Upon first thaw, separate Diluent 3 and Diluent 12 into aliquots appropriate for the size of your needs before refreezing. Prepare Blocker A Solution Follow the Blocker A instructions included in the kit. Prepare Standards MSD supplies calibrator for the Human TPO Kit at 20-fold higher concentration than the recommended highest standard. We recommend a 7-point standard curve with 3-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. Standard Human TPO (pg/mL) Dilution Factor Stock Calibrator 200 000 STD-01 10 000 20 STD-02 3333 3 STD-03 1111 3 STD-04 370 3 STD-05 123 3 STD-06 41 3 STD-07 14 3 STD-08 0 n/a To prepare 7 standard solutions plus a zero calibrator blank for up to 3 replicates: 1) Prepare the highest standard by adding 15 µL of stock calibrator to 285 µL of Diluent 12. Mix well. 2) Prepare the next standard by transferring 100 µL of the highest standard to 200 µL of Diluent 12. Mix well. Repeat 3-fold serial dilutions 5 additional times to generate 7 standards. 3) Use Diluent 12 as the blank. Dilute Samples For human serum and plasma samples, MSD recommends 2-fold dilution in Diluent 12; however, you may need to adjust the dilution factor for the sample set under investigation. To dilute sample 2-fold, add 75 µL of sample to 75 µL of Diluent 12. 18043-v1-2013Aug | 6 Prepare Detection Antibody Solution MSD provides detection antibody as a 50X stock solution. The working detection antibody solution is 1X. For 1 plate, combine: 60 µL of 50X SULFO-TAG Anti-hu TPO Antibody 2940 µL of Diluent 3 Prepare Read Buffer MSD provides Read Buffer T as a 4X stock solution. The working solution is 2X. For 1 plate, combine:
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