检测TNF的 elisa试剂盒说明书.

Quantikine?

Rat TNF-a Immunoassay

Catalog Number RTA00

SRTA00

PRTA00

For the quantitative determination of rat tumor necrosis factor alpha (TNF-a

concentrations in cell culture supernates, rat serum, and 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

Contents Page

INTRODUCTION2 PRINCIPLE OF THE

ASSAY. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3 LIMITATIONS OF THE

PROCEDURE3 PRECAUTION. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3

TECHNICAL HINTS3 MATERIALS

PROVIDED. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4 STORAGE5 OTHER

SUPPLIES REQUIRED. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5 SAMPLE

COLLECTION AND STORAGE6 SAMPLE

PREPARATION. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6 REAGENT

PREPARATION7 ASSAY

PROCEDURE. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .8 PROCEDURE

SUMMARY AND CHECKLIST9 CALCULATION OF

RESULTS. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .10 TYPICAL DATA10

PRECISION. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .11 RECOVERY11

LINEARITY. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .12 SENSITIVITY12

CALIBRATION. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .13 SAMPLE

VALUES13 SPECIFICITY. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .13

REFERENCES14 PLATE LAYOUT. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .15

MANUFACTURED AND DISTRIBUTED BY:

R&D Systems, ONE:(800 343-7475

614 McKinley Place NE(612 379-2956

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United States of America E-MAIL:info@

DISTRIBUTED BY:

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INTRODUCTION

Tumor necrosis factor alpha (TNF-a, also known as cachectin; and tumor necrosis

factor beta (TNF-b, also known as lymphotoxin, are two closely related proteins

(approximately 34% amino acid sequence identity that bind to the same cell surface

receptors and show many common biological functions. TNF-a and -b play critical roles

in normal host resistance to infection and to the growth of malignant tumors, serving as

immunostimulants and as mediators of the inflammatory response. Over- production of

TNFs, however, has been implicated as playing a role in a number of pathological

conditions, including cachexia, septic shock, and autoimmune disorders. TNF-a is

produced by activated macrophages and other cell types including T and B cells, NK

cells, LAK cells, astrocytes, endothelial cells, smooth muscle cells and some tumor cells

(1 - 4.

Rat TNF-a cDNA encodes a 235 amino acid (aa residue type II membrane protein (5.

The 156 aa residue soluble TNF-a is released from the C-terminus of the

membrane-anchored TNF-a by TNF-a-converting enzyme (TACE, a matrix

metalloprotease (6, 7. The membrane- anchored form of TNF-a has been shown to have

lytic activity and may also play an important role in intercellular communication (8. The

biologically active TNF-a has been shown to exist as a trimer (9, 10.

Two distinct TNF receptors, referred to as type I (or type B or p55 and type II (or

type A or p75, that specifically bind TNF-a and TNF-b with equal affinity have been

identified (11, 12. The two TNF receptors transduce signals independently of one another.

The amino acid sequence of the extracellular domains of the two receptors are

homologous and both receptors are members of the TNF receptor family which also

include the NGF receptor, fas antigen, CD27, CD30, and CD40. The intracellular

domains of the two receptors are apparently unrelated, suggesting that the two receptors

employ different signal transduction pathways. Soluble forms of both types of receptors

have been found in human serum and urine (13 - 15. These soluble receptors are capable

of neutralizing the biological activities of the TNFs and may serve to modulate the

activities of TNF.

The Quantikine Rat TNF-a Immunoassay is a 4.5 hour solid phase ELISA designed

to measure rat TNF-a levels in cell culture supernates, serum, and plasma. It contains

E. coli- expressed recombinant rat TNF-a and antibodies raised against the

recombinant factor. This immunoassay has been shown to quantitate the recombinant rat

TNF-a accurately. Results obtained using natural rat TNF-a showed dose response curves

that were parallel to the standard curves obtained using the recombinant kit standards.

These results indicate that the Quantikine Rat TNF-a Immunoassay kit can be used to

determine relative mass values for natural rat TNF-a.

PRINCIPLE OF THE ASSAY

This assay employs the quantitative sandwich enzyme immunoassay technique. A

monoclonal antibody specific for rat TNF-a has been pre-coated onto a microplate.

Standards, Control, and samples are pipetted into the wells and any rat TNF-a present is

bound by the immobilized antibody. After washing away any unbound substances, an

enzyme-linked polyclonal antibody specific for rat TNF-a 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 rat TNF-a 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, further dilute the

samples with Calibrator Diluent and repeat the assay.

·

Any variation in operator, pipetting technique, washing technique, incubation time

or

temperature, and kit age can cause variation in binding.

·

This assay is designed to eliminate interference by soluble receptors, binding

proteins, and other factors present in biological samples. Until all factors have been tested

in the Quantikine Immunoassay, the possibility of interference cannot be excluded.

PRECAUTION

The Stop Solution provided with this kit is an acid solution. Wear eye, hand, face,

and clothing protection when using this material.

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.

·

When using an automated plate washer, adding a 30 second soak period following

the addition of wash buffer, and/or rotating the plate 180 degrees between wash steps

may improve assay precision.

·

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 from light. Substrate Solution should change from colorless to

gradations of blue.

·

Stop Solution should be added to the plate in the same order as the Substrate

Solution.

The color developed in the wells will turn from blue to yellow upon addition of the

Stop Solution.

MATERIALS PROVIDED

Description Part #Cat. #

RTA00

Cat. #

SRTA00

Rat TNF-a Microplates- 96 well polystyrene microplates

(12 strips of 8 wells coated with a monoclonal antibody specific

for rat TNF-a.

890682 2 plates 6 plates

Rat TNF-a Conjugate- 23 mL/vial of a polyclonal antibody

against rat TNF-a conjugated to horseradish peroxidase with

preservatives.

892668 1 vial 3 vials

Rat TNF-a Standard- 1.6 ng/vial of recombinant rat TNF-a in a

buffered protein base with preservatives; lyophilized.890684 3 vials9 vials Rat TNF-

a Control- Recombinant rat TNF-a in a buffered

protein base with preservatives; lyophilized. The concentration

range of rat TNF-a after reconstitution is shown on the vial label.

The assay value of the Control should be within the range

specified on the label.

890685 3 vials9 vials

Assay Diluent RD1-41- 12.5 mL/vial of a buffered protein base

with preservatives.895514 1 vial 3 vials Calibrator Diluent RD5-17- 21 mL/vial of a

buffered protein

base with preservatives.895512 2 vials 6 vials Wash Buffer Concentrate- 50 mL/vial

of a 25-fold concentrated

solution of a buffered surfactant with preservative.895024 1 vial 3 vials Color

Reagent A- 12.5 mL/vial of stabilized hydrogen peroxide.895000 1 vial 3 vials Color

Reagent B- 12.5 mL/vial of stabilized chromogen

(tetramethylbenzidine.895001 1 vial 3 vials Stop Solution- 23 mL/vial of a diluted

hydrochloric acid solution.895174 1 vial 3 vials Plate Covers- Adhesive strips.___8

strips24 strips RTA00 contains sufficient materials to run ELISAs on two 96 well plates.

SRTA00 (SixPak contains sufficient materials to run ELISAs on six 96 well plates.

This kit is also available in a PharmPak (R&D Systems, Catalog # PRTA00.

PharmPaks contain sufficient materials to run ELISAs on 50 microplates. Specific vial

counts of each component may vary. Please refer to the literature accompanying your

order for specific vial counts.

*Provided this is within the expiration date of the kit.

OTHER SUPPLIES REQUIRED

·

Microplate reader capable of measuring absorbance at 450 nm, with the correction

wavelength set at 540 nm or 570 nm.

·

Pipettes and pipette tips.

·

Deionized or distilled water.

·

Squirt bottle, manifold dispenser, or automated microplate washer.

·

100 mL and 1000 mL graduated cylinders.

·

Polypropylene test tubes for dilution.

SAMPLE COLLECTION AND STORAGE

Cell Culture Supernates- Remove particulates by centrifugation and assay

immediately or aliquot and store samples at?

-20°

C. Avoid repeated freeze-thaw cycles.

Serum- Allow blood samples to clot for 2 hours at room temperature before

centrifuging for 20 minutes at 1000 x g. Remove serum and assay immediately or aliquot

and store samples at?

-20°

C. Avoid repeated freeze-thaw cycles.

Plasma- Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge for

20 minutes at 1000 x g within 30 minutes of collection. Assay immediately or

aliquot and store samples at?

-20°

C. Avoid repeated freeze-thaw cycles.

Note:Grossly hemolyzed or lipemic samples may not be suitable for measurement of

rat TNF-a with this assay.

SAMPLE PREPARATION