1、ELISA 试剂盒使用说明书(5 孔板格式)

ELISA kit

Instruction Manual

5-plate format

July, 2006

For research use only.

Not for use in diagnostic or therapeutic procedures.

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Contents

Introduction

Abbreviations

Contents of the kit

Hazard information

Materials and reagents required but not provided

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Working solutions

General procedure

Coating antibodies

Blocking

Test samples and standards

Biotinylated detector antibodies

SPP conjugate

Substrate

Cytokine standards

Storage kit reagents

Directions for washing

Trouble shooting

References

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Abbreviations

APC

BSA

CD

CSB

DMSO

ELISA

GM-CSF

IFN

Antigen presenting cells

Bovine serum albumin

Cluster of differentiation

Cytokine stabilization buffer

Dimethyl sulfoxide

Enzyme linked immunosorbent assay

Granulocyte-Macrophage Colony Stimulating Factor

Interferon

IL

MHC

OD

PB

PBS

PBST

PBST-B

SPP

T

h

TMB

TNF

Interleukin

Major histocompatibility complex

Optical density

Phosphate buffer

Phosphate buffered saline

PBS containing 0.05% Tween-20

PBST containing 0.5% bovine serum albumin

Streptavidin-HRP polymer

T helper subset

Tetramethylbenzidine

Tumor necrosis factor

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Introduction

Cytokines are a group of regulatory proteins critically involved in many physiological processes

such as immune recognition, cell differentiation and cell proliferation. They have been identified

in many vertebrate species and are produced by a variety of different cell types. Cytokines are

usually produced transiently and locally, acting in a paracrine or autocrine manner. They

interact with high affinity cell surface receptors specific for each cytokine or cytokine group and

are active at very low concentrations mostly in the picogram range.

It is well known now that the type of an antigen-specific immune response largely depends on

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the selection or preferential activation of defined CD

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T cell subsets (i.e. T

h

1 and T

h

2).

Activation of these subsets is characterized by the secretion of distinct patterns of cytokines.

T

h

1, but not T

h

2 cells, primarily secrete IL-2 and IFN-γ while T

h

2, but not T

h

1 cells, produce

IL-4, IL-5, IL-6, IL-10 and IL-13. Other cytokines, such as TNF-α and GM-CSF are produced by

both T

h

subsets. In addition, the production of IL-12 and IL-10, produced by antigen presenting

cells (APC) such as macrophages and dendritic cells, critically contributes to the preferential

expansion of T

h

1- or T

h

2-type of cells. For instance, early production of IL-12 is considered

essential for the development of T

h

1 cells. On the other hand, the absence or low

concentrations of IL-12 and IFN-γ in the early phase of an immune response and concomitant

production of IL-4 by cells of the mastcell/basophil lineage or T cells themselves is known to

favor the development of T

h

2 cells. In addition to their regulatory effects on T

h

subset

differentiation, the cytokines released by the two types of T

h

cells also produce distinct effector

functions. For instance, IL-4 and IFN-γ have differential or antagonistic activities on

immunoglobulin isotype selection or MHC class II expression. Therefore, the properties of an

immune response can be best studied by determining the amounts of cytokines produced by

the responding T cells and APC.

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Contents of the kit

Items

Coating antibodies

Cytokine standard

Biotinylated detector antibodies

SPP conjugate (Streptavidin-HRP polymer)

TMB substrate tablets

Substrate buffer capsules

BSA stock solution (10%)

Cytokine stabilization buffer (CSB)**

Tween-20

ELISA plates

Adhesive cover slips

Quantity

(5-plate format)

1 vial

5 vials

1 vial

1 vial

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2 vials (24 ml)

1 vial (5 ml)

1 vial (5 ml)

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Storage

conditions

4ºC (39ºF)

4ºC (39ºF)

4ºC (39ºF)

≤ -20ºC (-4°F)

4ºC (39ºF)

Rt*

4ºC (39ºF)

4ºC (39ºF)

Rt*

Rt*

Rt*

*

**

Room temperature

For serum and plasma samples only; see under “Test samples and standards”

Hazard information

TMB (tetramethylbenzidine) and sodium perborate (in substrate buffer)

are both irritating to eyes, respiratory system and skin.

In case of contact with eyes, rinse immediately with plenty of water and

seek medical advice.

Materials and reagents required but not provided

• PB stock: dissolve 96.0 g Na

2

HPO

4

.2H

2

O plus 17.5 g KH

2

PO

4

in 1.0 L distilled water and

adjust pH to 7.4

• Sterile distilled water

• H

2

SO

4

• Dimethyl sulfoxide (DMSO)

• Pipetting devices for the accurate delivery of volume required for the assay performance

• Plate washer: automated or manual (squirt bottle, manifold dispenser, etc)

• Reading device for microtiter-plate set to 370, 450 and/or 655 nm

Working solutions

• PBS: add 10 ml PB stock and 8.8 g NaCl to 1 L distilled water. Adjust pH to 7.4.

Alternatively, use commercially available liquid PBS from Invitrogen or other suppliers.

Do not use commercially available PBS tablets for the preparation of the coating

solution (the filler in the tablets interferes with the coating process).

• PBST: 0.5 ml Tween-20 dissolved in 1 L PBS.

• PBST-B: 2 ml BSA stock solution (10%) added to 38 ml PBST.

• Blocking buffer: 2 ml BSA stock solution (10%) added to 18 ml PBS (for 1 ELISA plate).

• Substrate buffer: the contents of one capsule is dissolved in 100 ml distilled water (takes

approximately 5 minutes). For optimal performance, the buffer solution should be used within

60 minutes.

• Stopping solution: 2 M H

2

SO

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General procedure

Coating antibodies

• Reconstitute the lyophilized antibodies by injecting 250 µl of sterile distilled water into the

vial. Mix the solution gently for approximately 15 seconds and allow it to stand for 2 minutes

at room temperature. Avoid vigorous shaking. To coat 96 wells of an ELISA plate 50 µl is

pipetted out of the vial (or use a frozen aliquot of 50 µl; see "Storage kit reagents") and

added to 5 ml PBS. Mix gently.

• Add 50 µl of diluted antibody solution to each well of the ELISA plate and fill up to 100 µl

with PBS.

• Seal the plate to prevent evaporation.

Incubate overnight at 4ºC or alternatively 1 to 2 hours at 37ºC.

Blocking

• Remove the coating antibody solution and wash the wells at least six times with PBST.

• Add 200 µl of blocking buffer.

• Seal the plate and incubate at 37ºC for 1 hour.

Test samples and standards

• Remove the blocking buffer but do not wash.

• Add 1/20 volume of CSB to serum or plasma samples but not to other samples such as cell

culture supernatants; CSB inhibits the degradation of cytokines in pure serum or plasma.

• Dilute standards and test samples in an appropriate diluent (see “Cytokine standards”).

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Add 100 µl to each well.

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Seal the plate and incubate at 37ºC for 2 hours or overnight at 4ºC.

Biotinylated detector antibodies

• Remove test samples/standards and wash the wells at least six times with PBST.

• Reconstitute the lyophilized antibodies by injecting 0.5 ml of sterile distilled water into the

vial. Mix the solution gently for approximately 15 seconds and allow it to stand for 2 minutes

at room temperature. Avoid vigorous shaking. Hundred microliter is pipetted out of the vial

(or use a frozen aliquot of 100 µl; see "Storage kit reagents") and added to 10 ml PBST-B.

Mix gently.

• Add 100 µl of diluted antibody solution to each well.

• Seal the plate and incubate at 37ºC for 1 hour.

SPP conjugate

• Remove detector antibody solution and wash the wells at least six times with PBST.

• Reconstitute the contents of the vial by injecting 0.5 ml of sterile distilled water into the vial.

Mix the solution gently for approximately 15 seconds and allow it to stand for 1 minute at

room temperature. Avoid vigorous shaking. Hundred microliter is pipetted out of the vial (or

use a frozen aliquot of 100 µl; see "Storage kit reagents") and added to 10 ml PBST-B. Mix

gently.

• Add 100 µl to each well.

• Seal the plate and incubate at 37ºC for 1 hour.

Substrate

• Remove SPP conjugate and wash the wells at least six times with PBST.

• Dissolve one TMB tablet in 1.0 ml DMSO (vortex at high speed for 5 minutes for complete

dissolution) and than add 10 ml substrate buffer.

• Mix thoroughly and immediately dispense 100 µl into each well. Leave the plate on the

laboratory bench at room temperature (color development between 10 and 30 minutes).

The substrate produces a soluble end-product that is blue in color and can be read

spectrophotometrically at 370 or 655 nm. The reaction can be stopped by adding 50 µl of

2 M H

2

SO

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(resulting in a yellow solution which can be read at 450 nm).

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Cytokine standards

For maximum recovery, the vial with lyophilized cytokine standard should be reconstituted in

0.5 ml distilled water and allowed to stand for 1 minute at room temperature. Thereafter, the

reconstituted cytokine standard (stock solution) is placed on melting ice and is immediately

diluted as indicated below (preferentially within one hour). Use vials with cytokine standards

only once.

Please note that temperature of buffers and standard solution(s) should now be kept at 0-4ºC

until use in the ELISA.

The total amount of cytokine standard is indicated on the label of the vial (ng/vial). After

reconstitution in 0.5 ml water, the concentration (ng/ml) will become twice the amount on the

label [e.g. amount on label is 4.8 ng/vial; after reconstitution, the concentration becomes

9.6 ng/ml = 9600 pg/ml].

The standard stock solution is diluted to 320 pg/ml in PBST-B (highest concentration cytokine to

be used in the standard range).

The linear region of the cytokine standard curve is now obtainable in a series of two-fold

dilutions in PBST-B ranging from 320 to 5 pg/ml. Always include a blank control (PBST-B only)

in the standard range.

Before establishing the standard curve, the OD value of the blank control () is subtracted

from the measured OD values of the different standard solutions. The standard curve is now

plotted as the standard cytokine concentration versus the corresponding (measured) OD value

minus . In addition, the actual OD values of the test samples are determined by

subtracting from the measured OD values.

The concentration of the cytokine in the test sample can then be interpolated from the standard

curve. It is useful to prepare a series of dilutions of the unknown test sample to assure that the

OD will fall in the linear portion of the standard curve.

Note 1: The OD value measured for the blank control () must be below 0.2.

Note 2: for measuring cytokines in cell culture supernatant, samples should be diluted in

PBST-B. However, when measuring cytokines in pure serum or plasma, the diluent for the

standard and blank control should preferentially be control serum or plasma originating from the

same species.

Storage kit reagents

The vials with lyophilized coating antibodies and biotinylated detector antibodies can be safely

stored in a refrigerator for a defined length of time (expiry date indicated on the vial). After

reconstitution, the antibodies remain fully active for minimal 6 months at 4ºC (39ºF) when kept

sterile. However, it is strongly recommended to divide the reconstituted antibody solutions into

small aliquots for single use. These aliquots should be stored at ≤ -20ºC. Under these

conditions the antibodies are stable for at least one year.

Upon arrival, the vial with lyophilized SPP conjugate should be stored at ≤ -20°C. Storage of the

vial at room temperature or at 4ºC for several months may lead to lower OD readings in the

ELISA. After reconstitution, the SPP solution is stable for 2 months at 4°C but rapidly looses

activity when stored at room temperature. It is strongly recommended that after reconstitution,

the solution is immediately divided into small aliquots for single use and stored at ≤ -20°C.

Under these conditions SPP is stable for minimal 12 months.

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Directions for washing

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Incomplete washing will adversely affect the assay. All washing must be performed with

wash buffer (PBST).

Washing can be performed manually as follows: completely aspirate the liquid from all wells

by gently lowering an aspiration tip (aspiration device) into each well. After aspiration, fill the

wells with at least 300 µl wash buffer. Let soak for 10 to 20 seconds, then aspirate the

liquid. Repeat as directed under "General procedure". After washing, the plate is inverted

and tapped dry on absorbent paper.

Alternatively, the wash buffer may be put into a squirt bottle. If a squirt bottle is used, flood

the plate with wash buffer, completely filling all wells. After washing, the plate is inverted

and tapped dry on absorbent paper.

If using an automated washing device, the operating instructions should carefully be

followed.

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Trouble shooting

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Poor consistency of replicates can be overcome by increasing the stringency of washes

particularly after the incubation step with detector antibody.

High values of the blank control (optical density > 0.2) can be overcome by shortening the

incubation time with the substrate solution or is caused by improper washing procedures.

Inconsistent replicates may be due to cross-contamination of wells by improper pipetting

procedures.

If no signal is observed in the wells with the standards

• try a new vial with cytokine standard

• check the pH of the substrate solution (between 5.0 and 5.5)

• verify whether the antibody, SPP conjugate and standard

preparations were properly diluted

Avoid sodium azide in wash buffers and diluents, as this is an inhibitor of peroxidase

activity.

Storage of reconstituted SPP at room temperature for several days can lead to a significant

loss of SPP activity and consequently low OD readings.

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References

Books:

• Practice and theory of enzyme immunoassays 1985

In: Laboratory techniques in biochemistry and molecular biology, Vol.15

(eds and P.H. van Knippenberg)

Science Publishers bv, Amsterdam, The Netherlands

• ELISA and other Solid Phase Immunoassays.

Theoretical and Practical Aspects 1988

(eds and combe)

John Wiley & Sons Ltd, Chichester, UK

• A practical guide to ELISA 1991

(ed ) Pergamon Press, Oxford, UK

Review of U-CyTech ELISA references:

Human cytokines:

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Monkey cytokines:

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Mouse cytokines:

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Rat cytokines:

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Dieleman, J.M. et al. 2006 Life Sci. 79: 551-558

Pacheco-López, G. et al. 2005 J. Neurosci. 25: 2330-2337

Sajti, E. et al. 2004 Brain Behav. Immun. 18: 505-514

Teunis, M.A.T. et al. 2002 J. Neuroimmun. 13: 30-38

Eijkelkamp, N. et al. 2004 J. Neuroimmun. 150: 3-9

Kavelaars, A. et al. 2005 J. Neuroimmun. 161: 162-168

Vroon, A. et al. 2005 J. Immunol. 174: 4400-4406

Fallon, P.G. et al. 2003 J. Infect. Dis. 187: 939-945

Hartman, G. et al. 2005 Vaccine 23: 3310-3317

Kornfeld, C. et al. 2005 J. Clin. Invest. 115: 1082-1091

Mascarell, L. et al. 2006 Vaccine 24: 3490-3499

Polakos, N.K. et al. 2001 J. Immunol. 166: 3589-3598

de Swart, R.L. et al. 2002 J. Virol. 76: 11561-11569

Arend, S.M. et al. 2000 J. Infect. Diseases 181: 1850-1854

Demirkiran, A. et al. 2006 Liver Transpl. 12: 277-284

Hoogendoorn, M. et al. 2005 Clin. Cancer Res. 11: 5310-5318

Tang, Y-M. et al. 2006 World J. Gastroenterol. 11: 4575-4578

de Waal, L. et al. 2004 J. Virol. 78: 1775-1781

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