Rat IFN-خ³ Immunoassay 2018. 8. 14.آ  Rat IFN-خ³ Control 890063 2 vials 12 vials Recombinant...

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Transcript of Rat IFN-خ³ Immunoassay 2018. 8. 14.آ  Rat IFN-خ³ Control 890063 2 vials 12 vials Recombinant...

  • Rat IFN-γ Immunoassay

    Quantikine® ELISA

    This package insert must be read in its entirety before using this product. For research use only. Not for use in diagnostic procedures.

    Catalog Number RIF00 Catalog Number SRIF00 Catalog Number PRIF00

    For the quantitative determination of rat Interferon gamma (IFN-γ) concentrations in cell culture supernates and serum.

  • 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: info@RnDSystems.com

    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: info@RnDSystems.co.uk

    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: info@RnDSystemsChina.com.cn

    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 ............................................................................................................................................4 PRECAUTIONS ........................................................................................................................................................................4 SAMPLE COLLECTION & STORAGE ................................................................................................................................4 REAGENT PREPARATION ....................................................................................................................................................5 ASSAY PROCEDURE ............................................................................................................................................................6 CALCULATION OF RESULTS ..............................................................................................................................................7 TYPICAL DATA ........................................................................................................................................................................7 PRECISION ...............................................................................................................................................................................8 RECOVERY................................................................................................................................................................................8 LINEARITY ................................................................................................................................................................................8 SENSITIVITY ............................................................................................................................................................................9 CALIBRATION .........................................................................................................................................................................9 SAMPLE VALUES ....................................................................................................................................................................9 SPECIFICITY .............................................................................................................................................................................9 REFERENCES ........................................................................................................................................................................ 10

  • www.RnDSystems.com 1

    INTRODUCTION Interferon-gamma (IFN-γ, also known as type II interferon) is an important immunoregulatory cytokine that was originally identified through its anti-viral activity (1, 2). It plays key roles in host defense by exerting antiviral, antiproliferative, and immunoregulatory activities (3, 4). On many cell types, IFN-γ induces the production of cytokines and upregulates the expression of various membrane proteins including class I and II MHC antigens, Fc receptors, leukocyte adhesion molecules, and B7 family antigens. IFN-γ is a potent activator of macrophage effector functions. It directs the synthesis, class switching, and secretion of immunoglobulins by B cells. IFN-γ also influences T-helper cell phenotype development by inhibiting Th2 differentiation and stimulating Th1 development (3, 4). IFN-γ plays a central role in the progression of inflammatory diseases such as autoimmunity and atherosclerosis (5, 6).

    Biologically active IFN-γ consists of a noncovalently linked homodimer of 20 - 25 kDa variably glycosylated subunits (7). Mature rat IFN-γ shares 86% amino acid (aa) sequence identity with mouse IFN-γ and 37% - 45% aa identity with bovine, canine, cotton rat, equine, feline, human, porcine, and rhesus IFN-γ. IFN-γ dimers bind to transmembrane IFN-γ RI (alpha subunits) which then interact with transmembrane IFN-γ RII (beta subunits) to form the functional receptor complex of two α and two β subunits (8, 9). Inclusion of IFN-γ RII in the receptor complex increases the ligand binding affinity as well as the efficiency of signal transduction (9, 10). Whereas the α-subunit is expressed constitutively on many cell types, the cellular regulation of the β-subunit correlates with an IFN-γ responsive state and is tightly regulated (8).

    IFN-γ is produced by a number of cell types under inflammatory conditions, including dendritic epidermal/γδ T cells (11), keratinocytes (12), peripheral blood γδ T cells (13), mast cells (14), neurons (15), CD8+ T cells (16), macrophages (17), B cells (18), neutrophils (19), NK cells (20), and CD4+ T cells (21).

    The Quantikine® Rat IFN-γ Immunoassay is a 4.5 hour solid phase ELISA designed to measure rat IFN-γ levels in cell culture supernates and serum. It contains E. coli-expressed recombinant rat IFN-γ and antibodies raised against the recombinant factor. This immunoassay has been shown to quantitate recombinant rat IFN-γ accurately. Results obtained using natural rat IFN-γ 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 rat IFN-γ.

  • For research use only. Not for use in diagnostic procedures.2

    PRINCIPLE OF THE ASSAY This assay employs the quantitative sandwich enzyme immunoassay technique. A polyclonal antibody specific for rat IFN-γ has been pre-coated onto a microplate. Standards, control, and samples are pipetted into the wells and any IFN-γ present is bound by the immobilized antibody. After washing away any unbound substances, an enzyme-linked polyclonal antibody specific for rat IFN-γ is added to the wells. Following a wash to remove any unbound antibody- enzyme reagent, a substrate solution is added to the wells. The enzyme reaction yields a blue product that turns yellow when the Stop Solution is added. The intensity of the color measured is in proportion to the amount of IFN-γ bound in the initial step. The sample values are then read off the standard curve.

    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 generate values higher than the highest standard, dilute the samples with calibrator diluent and repeat the assay.

    • Any variation in diluent, operator, pipetting technique, washing technique, incubation time or temperature, and kit age can cause variation in binding.

    • Variations in sample collection, processing, and storage may cause sample value differences.

    • This assay is designed to eliminate interference by other factors present in biological samples. Until all factors have been tested in the Quantikine® Immunoassay, the possibility of interference cannot be excluded.

    TECHNICAL HINTS • When mixing or reconstituting protein solutions, always avoid foaming.

    • To avoid cross-contamination, change pipette tips between additions of each standard level, between sample additions, and between reagent additions. Also, use separate reservoirs for each reagent.

    • For best results, pipette reagents and samples into the center of each well.

    • It is recommended that the samples be pipetted within 15 minutes.

    • To ensure accurate results, proper adhesion of plate sealers during incubation steps is necessary.

    • Substrate Solution should remain colorless until added to the plate. Keep Substrate Solution protected