Valukine™ ELISA Kit
Human IL-2 Immunoassay
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Catalog Number：VAL110
For the quantitative determination of human interleukin 2 (IL-2) concentrations in cell culture supernates.
This kit contains sufficient materials to run an ELISA on one 96 well plate. This package inrt must be read in its entirety before using this product. FOR RESEARCH USE ONLY.
NOT FOR USE IN DIAGNOSTIC PROCEDURES.
TABLE OF CONTENTS
Contents Page INTRODUCTION (3)
PRINCIPLE OF THE ASSAY (3)
LIMITATIONS OF THE PROCEDURE (3)
MATERIALS PROVIDED (4)
STORAGE (4)
OTHER SUPPLIES REQUIRED (4)
PRECAUTION (4)
SAMPLE COLLECTION AND STORAGE (4)
REAGENT PREPARATION (5)
ASSAY PROCEDURE (6)
CALCULATION OF RESULTS (6)取得进步
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TYPICAL DATA (7)
PRECISION (7)
RECOVERY (7)
SENSITIVITY (7)
CALIBRATION (7)
LINEARITY (8)
SAMPLE VALUES (8)
edasSPECIFICITY (8)
REFERENCES (9)
PLATE LAYOUT (10)
MANUFACTURED BY:
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R&D Systems, Inc.
614 McKinley Place NE
Minneapolis, MN 55413
United States of America
DISTRIBUTED BY:
R&D Systems China Co. Ltd.
24A1 Hua Min Empire Plaza TELEPHONE: +86 (21) 52380373
726 West Yan An Road FAX: +86 (21) 52371001
Shanghai PRC 200050 E-MAIL: ************************
INTRODUCTION
bateHuman Interleukin 2 (IL-2), also known as T cell growth factor (TCGF), is a 15-18 kDa variably glycosylated α-helical polypeptide that is a member of the common gamma chain (γc) cytokine family (1-4). It exists as a monomer and has a notably short half-life (< 30 minutes) (1). Human IL-2 is synthesized as a 153 amino acid (aa) precursor that contains a 20 aa signal quence plus a 133 aa mature region (5, 6). The mature protein contains one utilized O-linked glycosylation site at Thr3, plus three cysteines, two of which form an intrachain disulfide bond that is esntial for activity (7). M
ature human IL-2 shares 73%, 66%, 78% and 97% aa quence identity with canine, rat, feline and rhesus monkey IL-2, respectively. Although human IL-2 shares only approximately 60% aa identity with the highly polymorphic mou IL-2, human IL-2 is known to be active on mou IL-2 responsive cells. Cells reported to crete IL-2 include γδT cells (8), activated conventional CD4+ and CD8+ T cells (1, 9), neurons (10, 11), microglia (12), and hematopoietic stem cells (13).
The receptor for IL-2 (IL-2R) is compod of three subunits, the 55 kDa CD25/IL-2Rαchain, the
70 kDa CD122/IL-2Rβchain, and the 65 kDa CD132/γc chain (1, 3). IL-2 first binds to CD25, the binary complex then recruits CD122 and CD132 to form the quaternary signaling complex (1, 14). In addition to IL-2, CD122/IL-2Rβis ud by IL-15 in its quaternary signaling complex. CD132/γc also rves as a signaling receptor for IL-4, -7, -9, -15, and -21 (1, 3).
In vitro studies have shown an important role for IL-2 in T cell activation and expansion. In vivo, IL-2 is critical for the development, maintenance and function of regulatory T cells (Treg) which provide protection against autoimmune dia. On the other hand, IL-2 can also promote autoimmune inflammation in target organs through its roles in regulating the expression of T cell trafficking genes, and production of Th2 cytokines. Within the CD8+ T cell subt, IL-2 is esntial for optimal primary r
espons and differentiation into terminal effector cells. IL-2 also promotes the development of activated CD8+ T cells into memory cells. (1).
PRINCIPLE OF THE ASSAY
This assay employs the quantitative sandwich enzyme immunoassay technique. A monoclonal antibody specific for IL-2 has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and any IL-2 prent is bound by the immobilized antibody. After washing away any unbound substances, an enzyme-linked polyclonal antibody specific for IL-2 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 IL-2 bound in the initial step. The color development is stopped and the intensity of the color is measured.
LIMITATIONS OF THE PROCEDURE
w FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES.
w The kit should not be ud beyond the expiration date on the kit label.
w Do not mix or substitute reagents with tho from other lots or sources.
discretew If samples generate values higher than the highest standard, dilute the samples with Diluent and repeat the assay.
w Any variation in operator, pipetting technique, washing technique, incubation time or temperature, and kit age can cau variation in binding.
MATERIALS PROVIDED
IL-2 Microplate - 96 well polystyrene microplate (12 strips of 8 wells) coated with a mou monoclonal antibody against human IL-2.
IL-2 Conjugate - 21 mL of polyclonal antibody against IL-2 conjugated to horradish peroxida with prervatives.
IL-2 Standard - 10 ng of recombinant human IL-2 in a buffered protein ba with prervatives; lyophilized.
Diluent Concentrate 5X - 21 mL of a 5X concentrated buffered protein ba with prervatives.
Wash Solution Concentrate 25X - 21 mL of a 25X concentrated solution of buffered surfactant with prervatives.
Substrate A - 12.5 mL of stabilized hydrogen peroxide.
Substrate B - 12.5 mL of stabilized chromogen (tetramethylbenzidine).
Stop Solution 1 - 6 mL of 2 N sulfuric acid.
Plate Covers - 3 adhesive strips.
STORAGE
Unopened Kit Store at 2-8° C. Do not u past kit expiration date.
Opened/ Reconstituted Reagents Diluted Wash Solution
May be stored for up to 1 month at 2-8° C.*
Stop Solution 1
Diluent 1X
Conjugate
Unmixed Substrate A
Unmixed Substrate B
Standard
Aliquot and store for up to 1 month at -20° C in a manual
defrost freezer.* Avoid repeated freeze-thaw cycles. Microplate Wells
Return unud wells to the foil pouch containing the
desiccant pack, real along entire edge of zip-al. May
金山 在线翻译be stored for up to 1 month at 2-8° C.*
*Provided this is within the expiration date of the kit.
OTHER SUPPLIES REQUIRED
w Microplate reader capable of measuring absorbance at 450 nm, with the correction wavelength t at 540 nm or 570 nm.
w Pipettes and pipette tips.
w Deionized or distilled water.
w Squirt bottle, manifold dispenr, or automated microplate washer.
w500 mL graduated cylinder.
PRECAUTION
The Stop solution 1 provided with this kit is an acid solution. Wear eye, hand, face, and clothing protection when using this material.
SAMPLE COLLECTION AND STORAGE
Cell Culture Supernates - Remove particulates by centrifugation and assay immediately or aliquot and store samples at ≤-20℃. Avoid repeated freeze-thaw cycles. Samples may require dilution with Diluent 1X.
REAGENT PREPARATION
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Bring all reagents to room temperature before u.
pirateWash Solution - If crystals have formed in the concentrate, warm to room temperature and mix gently until the crystals have completely dissolved. Dilute 20 mL of Wash Solution Concentrate into deionized or distilled water to prepare 500 mL of Wash Buffer.
Substrate Solution - Substrates A and B should be mixed together in equal volumes within 15 minutes of u. Protect from light. 200 μL of the resultant mixture is required per well.
Diluent 1X –Add 20 mL of Diluent Concentrate 5X into 80 mL of deionized or distilled water to prepare 100 mL of Diluent 1X.
IL-2 S tandard - Reconstitute the IL-2 Standard with 5 mL of Diluent 1X. This reconstitution produces a stock solution of 2000 pg/mL. Allow the standard to sit for a minimum of 15 minutes with gentle agitation prior to making dilutions.
Pipette 500 μL of Diluent 1X into each tube.U the stock solution to produce a dilution ries (below). Mix each tube thoroughly before the next transfer. The undiluted standard rves as the high standard (2000 pg/mL). The Diluent 1X rves as the zero standard (0 pg/mL).
