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Human Thrombopoietin Quantikine Quantikine® ELISA Human Thrombopoietin Immunoassay Catalog Number DTP00B For the quantitative determination of human Thrombopoietin (Tpo) concentrations in cell culture supernates, serum, and platelet-poor plasma. This package insert must be read in its entirety before using this product. For research use only. Not for use in diagnostic procedures. TABLE OF CONTENTS SECTION PAGE INTRODUCTION ....................................................................................................................................................................1 PRINCIPLE OF THE ASSAY ..................................................................................................................................................2 LIMITATIONS OF THE PROCEDURE ................................................................................................................................2 TECHNICAL HINTS ................................................................................................................................................................2 MATERIALS PROVIDED & STORAGE CONDITIONS ..................................................................................................3 OTHER SUPPLIES REQUIRED ............................................................................................................................................3 PRECAUTIONS ........................................................................................................................................................................4 SAMPLE COLLECTION & STORAGE ................................................................................................................................4 REAGENT PREPARATION ....................................................................................................................................................5 ASSAY PROCEDURE ............................................................................................................................................................6 CALCULATION OF RESULTS ..............................................................................................................................................7 TYPICAL DATA ........................................................................................................................................................................7 PRECISION ...............................................................................................................................................................................8 RECOVERY................................................................................................................................................................................8 LINEARITY ................................................................................................................................................................................9 SENSITIVITY ............................................................................................................................................................................9 CALIBRATION .........................................................................................................................................................................9 SAMPLE VALUES ................................................................................................................................................................. 10 SPECIFICITY .......................................................................................................................................................................... 10 REFERENCES ........................................................................................................................................................................ 11 PLATE LAYOUT .................................................................................................................................................................... 12 MANUFACTURED AND DISTRIBUTED BY: USA & Canada | R&D Systems, Inc. 614 McKinley Place NE, Minneapolis, MN 55413, USA TEL: (800) 343-7475 (612) 379-2956 FAX: (612) 656-4400 E-MAIL: [email protected] DISTRIBUTED BY: UK & Europe | R&D Systems Europe, Ltd. 19 Barton Lane, Abingdon Science Park, Abingdon OX14 3NB, UK TEL: +44 (0)1235 529449 FAX: +44 (0)1235 533420 E-MAIL: [email protected] China | R&D Systems China Co., Ltd. 24A1 Hua Min Empire Plaza, 726 West Yan An Road, Shanghai PRC 200050 TEL: +86 (21) 52380373 FAX: +86 (21) 52371001 E-MAIL: [email protected] INTRODUCTION Human thrombopoietin (Tpo), also known as megakaryocyte growth and development factor (MGDF), Mpl-ligand, and megakaryocyte colony stimulating factor (MK-CSF), is a 60-70 kDa, 332 amino acid (aa) residue glycosylated polypeptide that is believed to play a key role in the development of megakaryocytes (1-4). Secreted principally by hepatocytes (5) and bone marrow stromal cells (6), the Tpo molecule can be structurally and functionally divided into N- and C-terminal halves. The N-terminal half of Tpo is structurally analogous to the erythropoietin (Epo) molecule, showing approximately 25% aa residue identity and conservation of a number of cysteines. Functionally, this area is suggested to be responsible for receptor binding (1, 2, 7). The C-terminal region, on the other hand, is structurally characterized by a high percentage of serine and proline residues and contains a number of potential N-linked glycosylation sites. The C-terminus is believed to contribute to overall molecule stability and circulating half-life (1, 2, 8). Across species, there is marked conservation of aa residues, with human Tpo demonstrating 75%, 73% and 76% aa identity with rat (9), mouse (2) and dog (2) Tpo, respectively. The conservation of aa structure accounts for the cross-species bioactivity noted on human and mouse cells. An 84 kDa receptor for Tpo, Mpl, has been identified in both humans and mice (12-14). In humans, two forms of the receptor have been noted, the longest of which is 607 aa residues in length, with a 463 aa extracellular region, a 22 aa transmembrane segment, and a 122 aa cytoplasmic domain rich in proline and serine (12). The shorter receptor form differs from the longer form only in that the cytoplasmic domain is only 66 amino acids in length with a high proline content (12). The extracellular region can be divided into two subdomains. The N-terminal subdomain contains the Tpo binding site and four conserved cysteines, characteristic of the hematopoietin receptor superfamily. The juxtamembrane subdomain shows a WS-X-WS box (specifically, WS-S-WS), also characteristic of members of the hematopoietin receptor superfamily (12). Relative to the human, the mouse receptor is almost identical in length (608 aa residues) and shows 81% overall aa identity, 78% in the extracellular region (13). Both the human and the mouse c-mpl gene show a possibility for an alternative splice event that could produce a soluble form of the receptor (13, 14). Cells known to express Mpl include platelets, megakaryocytes and CD34+CD61+CD41a+ megakaryocyte progenitor cells (15), BFU-E cells (16), plus the HEL erythroid (12), MO-7E (15), and CMK (17) cell lines. Functionally, Tpo seems to increase the absolute number of megakaryoctes (18), to increase megakaryocyte viability (19), and to shorten the time necessary for cell cycling (18). Tpo does not seem to contribute to pro-platelet formation (19, 21). Circulating Tpo has been measured in both normal and pathophysiological conditions, including idiopathic thrombocytopenia purpura (ITP) (22-24), essential thrombocythemia (ET) (25), liver cirrhosis (26), amegakaryocytic thrombocytopenia (AMT) (27), and aplastic anemia (AA) (22-25). While Tpo levels may be regulated by megakaryocyte counts, and platelets may play a role in regulating plasma Tpo levels, there does not seem to be a direct correlation between platelet count and blood Tpo levels (22, 24, 26-28). The Quantikine® Human Thrombopoietin Immunoassay is a 4.5 hour solid phase ELISA designed to measure human Tpo in cell culture supernates, serum, and platelet-poor plasma. It contains NS0-expressed recombinant human Tpo and antibodies raised against the recombinant factor. Results obtained using natural human Tpo showed dose-response curves that were parallel to the standard curves obtained using the recombinant Quantikine® kit standards. These results indicate that this kit can be used to determine relative mass values for natural human Tpo. www.RnDSystems.com 1 PRINCIPLE OF THE ASSAY This assay employs the quantitative sandwich enzyme immunoassay technique. A monoclonal antibody specific for human Tpo has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and any Tpo present is bound by the immobilized antibody. After washing away any unbound substances, an enzyme-linked polyclonal antibody specific for human Tpo is added to the wells. Following a wash to remove any unbound antibody- enzyme reagent, a substrate solution is added to the wells and color develops in proportion to the amount of Tpo bound in the initial step. The color development is stopped and the intensity of the color is measured. LIMITATIONS OF THE PROCEDURE • FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES. • The kit should not be used beyond the expiration date on the kit label. • Do not mix or substitute reagents with those from other lots or sources. • If samples
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