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Human CCL5/RANTES Quantikine ELISA

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Human CCL5/RANTES Quantikine ELISA Quantikine™ ELISA Human CCL5/RANTES Immunoassay Catalog Number DRN00B Catalog Number SRN00B Catalog Number PDRN00B For the quantitative determination of human Regulated upon Activation, Normal T cell Expressed and presumably Secreted (RANTES) concentrations in cell culture supernates, serum, platelet-poor plasma, and urine. 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 PHARMPAK CONTENTS ......................................................................................................................................................4 OTHER SUPPLIES REQUIRED ............................................................................................................................................5 PRECAUTIONS ........................................................................................................................................................................5 SAMPLE COLLECTION & STORAGE ................................................................................................................................6 SAMPLE PREPARATION.......................................................................................................................................................6 REAGENT PREPARATION ....................................................................................................................................................7 ASSAY PROCEDURE ............................................................................................................................................................8 CALCULATION OF RESULTS ..............................................................................................................................................9 TYPICAL DATA ........................................................................................................................................................................9 PRECISION ............................................................................................................................................................................ 10 RECOVERY............................................................................................................................................................................. 10 LINEARITY ............................................................................................................................................................................. 11 SENSITIVITY ......................................................................................................................................................................... 11 CALIBRATION ...................................................................................................................................................................... 11 SAMPLE VALUES ................................................................................................................................................................. 12 SPECIFICITY .......................................................................................................................................................................... 13 REFERENCES ........................................................................................................................................................................ 14 Manufactured and Distributed by: USA R&D Systems, Inc. 614 McKinley Place NE, Minneapolis, MN 55413 TEL: 800 343 7475 612 379 2956 FAX: 612 656 4400 E-MAIL: [email protected] Distributed by: Europe | Middle East | Africa Bio-Techne Ltd. China Bio-Techne China Co., Ltd. 19 Barton Lane, Abingdon Science Park Unit 1901, Tower 3, Raffles City Changning Office, Abingdon OX14 3NB, UK 1193 Changning Road, Shanghai PRC 200051 TEL: +44 (0)1235 529449 TEL: +86 (21) 52380373 (400) 821-3475 FAX: +44 (0)1235 533420 FAX: +86 (21) 52371001 E-MAIL: [email protected] E-MAIL: [email protected] INTRODUCTION RANTES (Regulated upon Activation, Normal T cell Expressed and presumably Secreted), also known as CCL5, is a member of the "CC" subfamily of chemokines. It plays a primary role in the inflammatory immune response via its ability to chemoattract leukocytes and modulate their function. The cDNA for RANTES was initially discovered by subtractive hybridization as a T cell specific sequence (1, 2). Human RANTES cDNA encodes a highly basic 91 amino acid (aa) residue precursor polypeptide with a 23 aa hydrophobic signal peptide that is cleaved to generate the 68 aa mature protein (1, 2). Human RANTES exhibits approximately 85% homology with mouse RANTES at the deduced aa level (3, 4). RANTES is a potent chemoattractant for a number of different cell types including unstimulated CD4+/CD45RO+ memory T cells and stimulated CD4+ and CD8+ T cells with naive and memory phenotypes, NK cells, basophils, eosinophils, dendritic cells, mast cells, monocytes, and microglia (5-13). In addition to its effects on migration, RANTES can activate a number of cell types including T cells (14-16), monocytes (17), neutrophils (17), NK cells (7), dendritic cells (18), and astrocytes (19). T cell activation generally requires relatively high RANTES concentrations (~ 1 μM) and is dependent upon aggregation of the molecule and association with cell surface glycosaminoglycans (GAGs) (15-17). Whether this activity occurs in vivo remains unclear although in mice, intraperitoneally injected RANTES mutants that are unable to aggregate and/or bind GAG, are not capable of attracting leukocytes when compared to wild-type controls (20). Other in vivo studies show that RANTES knockout mice exhibit deficient recruitment of leukocytes to sites of acute inflammation (21). RANTES, is known to interact with four identified seven transmembrane G-protein coupled receptors: CCR1, CCR3, CCR4, and CCR5 (22-25). RANTES stimulation can initiate a variety of signaling cascades that are cell context dependent. For instance, in T-cells, RANTES can stimulate elevations of intracellular Ca2+ (26), and activation of focal adhesion kinase (FAK) (27), protein kinase A (28), PI3-kinase (14), Rho GTPase (29), and JAK/STAT signaling pathways (30). The cytomegalovirus protein US28 exhibits significant homology with CC chemokine receptors and is capable of binding RANTES (31). Membrane-spanning US28 can, depending on the context, signal in a constitutive manner (32), bind RANTES and initiate G-protein-mediated signaling cascades (33), or sequester RANTES and potentially alter inflammatory responses (34-36). The RANTES receptor CCR5 is also the primary co-receptor for R5 (M-tropic) variants of HIV-1 (37, 38). It has been demonstrated that RANTES, as well as the other CCR5 ligands, macrophage inflammatory protein (MIP)-1α and MIP-1β, can competitively inhibit CCR5/HIV-1 interaction and suppress viral infection in vitro (39, 40). These effects apparently do not require fully intact signaling from the CCR5 receptor (41). Consequently, modified forms of RANTES and non-peptide compounds that block the interaction of HIV-1 with CCR5 show promise for future therapies (41-44). In contrast, several reports show that RANTES can enhance in vitro replication of X4 (T-tropic) variants of HIV-1 that use CXCR4 as a co-receptor rather than CCR5 (45, 46). This activity usually requires relatively high RANTES concentrations (~μM) and is dependent upon interaction with cell surface GAGs, oligomerization, and activation of tyrosine kinase and MAP kinase signaling cascades (46, 47). The Quantikine™ Human CCL5/RANTES Immunoassay is a 3.5 hour solid phase ELISA designed to measure human RANTES levels in cell culture supernates, serum, platelet-poor plasma, and urine. It contains E. coli-expressed recombinant human RANTES and antibodies raised against the recombinant factor. This immunoassay has been shown to accurately quantitate the recombinant factor. Results obtained using natural human RANTES showed linear curves that were parallel to the standard curves obtained using the Quantikine standards. These results indicate that this kit can be used to determine relative mass values of human RANTES. www.RnDSystems.com 1 PRINCIPLE OF THE ASSAY This assay employs the quantitative sandwich enzyme immunoassay technique. A monoclonal antibody specific for human RANTES has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and any RANTES present is bound by the immobilized antibody. After washing away any unbound substances, an enzyme-linked polyclonal antibody specific for human RANTES is added to the wells. Following a wash to remove
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