PRODUCT INFORMATION & MANUAL 2018. 3. 23.آ  PRODUCT INFORMATION & MANUAL . Mouse/Rat TNF-خ±...

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Transcript of PRODUCT INFORMATION & MANUAL 2018. 3. 23.آ  PRODUCT INFORMATION & MANUAL . Mouse/Rat TNF-خ±...

  • PRODUCT INFORMATION & MANUAL

    Mouse/Rat TNF-α ValukineTM ELISA

    VAL609

    For the detection of mouse/rat TNF-α

    For research use only. Not for diagnostic or therapeutic procedures.

    Bio-Techne China Co. Ltd P: +86 (21) 52380373 P: 8009881270 F: +86 (21) 52381001

    info.cn@bio-techne.com

    Novus kits are guaranteed for 3 months from date of receipt

    mailto:info.cn@bio-techne.com

  • Tel: 800-988-1270 Email: info.cn@bio-techne.com 1 Web Site: www.novusbio.com/china

    TABLE OF CONTENTS I. BACKGROUND .................................................................................................. 2

    II. OVERVIEW ........................................................................................................ 2

    III. ADVANTAGES ................................................................................................... 3

    IV. EXPERIMENT .................................................................................................... 4

    V. KIT COMPONENTS AND STORAGE ................................................................ 5

    VI. PREPARATION .................................................................................................. 6

    VII. ASSAY PROCEDURE ........................................................................................ 7

    VIII. RERERENCES .................................................................................................. 9

    IX. TROUBLESHOOTING ..................................................................................... 10

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    I. BACKGROUND Tumor necrosis factor alpha (TNF-α), also known as cachectin and TNFSF1A, is the prototypic ligand of the TNF superfamily (1). It is a pleiotropic molecule that plays a central role in inflammation, immune system development, apoptosis, and lipid metabolism (2-5). TNF-α is also involved in a number of pathological conditions including asthma, Crohn’s diease, rheumatoid arthritis, neuropathic pain, obesity, type 2 diabetes, septic shock, autoimmunity, and cancer (5-11).

    Mouse TNF-α is synthesized as a 26 kDa type II transmembrane protein that consists of a 35 amino acid (aa) cytoplasmic domain, a 21 aa trasmembrane segment, and a 179 aa extracellular domain(ECD) (12). Within the ECD mouse TNF-α shares 95% aa sequence identity with rat, and 80% aa identitiy with canine, equine, feline, human, rabbit, andporcine TNF-α. It is produced by a wide variety of immune, epithelial, endothelial, and tumor cells. TNF-α is assembled intracellularly to form a nonconvalently linked homotrimer which is expressed on the cell surface (13). Cell surface TNF-α can both induce the lysis of tumor cells and virus infected cells, and generate its own downstream cell signaling following ligation by soluble TNF RI (14,15). Shedding of membrance bound TNF-α by TACE/ADAM17 releases the bioactive cytokine, a 55 kDa soluble trimer containing the TNF-α extracellular domain (16-18).

    TNF-α binds the ubiquitous 55-66 kDa TNF RI (19,20) and the hematopoietic cell restricted 78-80 kDa TNF RII (21,22), both of which are also expressed as hootrimers (1,23). Both type I and type II receptors bind TNF-α with comparable affinity and can promote NFκB activation (25-28). Only TNF RI, however, contains a cytoplasmic death domain which triggers the activation of apoptosis (3,28). Soluble forms of both types of receptors are released into human serum and urine and can neutralized the biological activity of TNF-α (29-31).

    II. OVERVIEW A. PRINCIPLE OF THE ASSAY

    This assay employs the quantitative sandwich enzyme immunoassay technique. Monoclonal antibody specific for mouse/rat TNF-α has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and any TNF-α present is bound by the immobilized antibody. After washing away any unbound substances, an enzyme-linked polyclonal antibody specific for mouse/rat TNF-α 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 mouse/rat TNF-α bound in the initial step. The color development is stopped and the intensity of the color is measured.

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    B. 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 Diluent 1× and repeat the assay. • Any variation in operator, pipetting technique, washing technique, incubation time

    or temperature, and kit age can cause variation in binding.

    III. ADVANTAGES A. PRECISION Intra-assay Precision (Precision within an assay) Two samples were tested twenty times on one plate to assess intra-assay precision. Inter-assay Precision (Precision between assays) Three samples were tested in twenty separate assays to assess inter-assay precision.

    Intra-assay Precision Inter-assay Precision

    Sample 1 2 1 2 3 n 20 20 20 20 20

    Mean (pg/mL) 54.6 116 405 45.0 111 Standard Deviation 1.5 4.5 12.5 4.27 7.3

    CV% 2.7 3.9 3.1 9.5 6.6

    B. RECOVERY

    The recovery of mouse TNF-α spiked to different levels in cell culture media was evaluated. The mouse sample recovery ranged from 88-107% with an average of 94%. The rat sample recovery ranged from 84-110% with an average of 95%.

    C. SENSITIVITY

    The minimum detectable dose (MDD) of mouse TNF-α is typically less than 5.5 pg/mL.

    The MDD was determined by adding two standard deviations to the mean optical density value of twenty zero standard replicates and calculating the corresponding concentration.

    D. CALIBRATION This immunoassay is calibrated against highly purified E. coli-expressed recombinant mouse TNF-α produced at R&D Systems.

    E. LINEARITY To assess the linearity of the assay, six cell culture media samples were spiked with high concentrations of mouse TNF-α and diluted with Diluent 1× to produce samples with values within the dynamic range of the assay.

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    Mouse samples Rat Samples

    Dilution Average % of Range (%) Average % of Range 1:2 97 94-103 94 91-97 1:4 105 99-111 90 89-91 1:8 106 97-114 98 -

    1:16 107 104-111 - -

    F. SAMPLE VALUES Cell Culture Supernates - Mouse thymoma cells (EL-4; 2×105 cells/mL) were cultured for 4 days in DMEM supplemented with 10% fetal calf serum and stimulated with 10 µg/mL LPS and 100 ng/mL recombinant mouse IL-10. An aliquot of the cell culture supernate was removed, assayed for levels of natural mouse TNF-α and measured 1220 pg/ml.

    Rat spleen tissue was homogenized and cells were cultured in RPMI media supplemented with 10% fetal bovine serum and stimulated with 10 µg/mL Con A for 18 hours. An aliquot of the cell culture supernate was tested for rat TNF-α and measured 110 pg/ml.

    G. SPECIFICITY This assay recognizes both natural and recombinant mouse TNF-α. The following factors were prepared at 100 ng/mL and assayed for cross-reactivity. Preparations of the following factors at 100 ng/mL in a mid-range rmTNF-α control were assayed for interference. No significant cross-reactivity or interference was observed. Recombinant mouse: Other Recombinant: CD40 RANK Canine TNF-α

    CD40 ligand Fas Fas ligand

    RANK ligand TRAIL TROY

    Human TNF-α Porcine TNF-α

    LIF TNF-β OPG

    40% cross-reactivity was observed with recombinant rat TNF-α. Recombinant mouse TNF RII ad recombinant mouse TNF RII were found to interfere in this assay at concentrations above 1.25 ng/mL and 12.5 ng/mL, respectively.

    IV. EXPERIMENT EXAMPLE STANDARD The standard curve is provided for demonstration only. A standard curve should be generated for each set of samples assayed.

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    V. KIT COMPONENTS AND STORAGE A. MATERIALS PROVIDED

    Parts Description Size Mouse TNF-α Microplate

    96 well polystyrene microplate (12 strips of 8 wells) coated with monoclonal antibody against mouse TNF-α

    1 plate

    Mouse TNF-α Conjugate

    21mL/vial of a polyclonal antibody against mouse TNF-α conjugated to horseradish peroxidase with preservatives

    1 vial

    Mouse TNF-α Standard

    Recombinant mouse TNF-α in a buffered protein base with preservatives; lyophilized.

    2 vial

    Calibrator Diluent RD5P(5×)

    21 mL/vial of a 5× concentrated buffered protein base with preservatives. For cell culture supernate samples.

    1 vial

    Wash Buffer Concentrate(25×)

    21 mL/vial of a 25× concentrated solution of buffered surfactant with preservatives.

    1 vial

    Color Reagent A 12.5 mL/vial of stabilized hydrogen peroxid