Human interferon γ(IFN-γ) antibody ELISA Kit说明书

Human interferon γ(IFN-γ) antibody

ELISA Kit

Catalog Number. CSB-E14210h

For the qualitative determination of human interferon γ(IFN-γ) antibody

concentrations in serum, plasma.

This package insert must be read in its entirety before using this product.

If You Have Problems

Technical Service Contact information

Phone: 86-27-87582341

Fax: 86-27-87196150

Email:****************

Web:

In order to obtain higher efficiency service, please ready to supply the lot number

of the kit to us (found on the outside of the box).

1

PRINCIPLE OF THE ASSAY

This assay employs the qualitative enzyme immunoassay technique.

The microtiter plate provided in this kit has been pre-coated with antigen.

Samples are pipetted into the wells with anti-human immunoglobulin conjugated

Horseradish Peroxidase (HRP). Any antibodies specific for the antigen present

will bind to the pre-coated antigen. Following a wash to remove any unbound

reagent, a substrate solution is added to the wells and color develops in

proportion to the amount of human IFN-γ antibody bound in the initial step. The

color development is stopped and the intensity of the color is measured.

SPECIFICITY

This assay has high sensitivity and excellent specificity for detection of human

IFN-γ antibody. No significant cross-reactivity or interference between human

IFN-γ antibody and analogues was observed.

Note: Limited by current skills and knowledge, it is impossible for us to complete

the cross-reactivity detection between human IFN-γ antibody and all the

analogues, therefore, cross reaction may still exist.

PRECISION

Intra-assay Precision (Precision within an assay): CV%<15%

Three samples of known concentration were tested twenty times on one plate to

assess.

Inter-assay Precision (Precision between assays): CV%<15%

Three samples of known concentration were tested in twenty assays to assess.

2

LIMITATIONS OF THE PROCEDURE

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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.

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 Immunoassay, the possibility of

interference cannot be excluded.

MATERIALS PROVIDED

Reagents

Coated assay plate

Negative Control

Positive Control

HRP-conjugate(100 x concentrate)

HRP-conjugate Diluent

Sample Diluent

Wash Buffer (25 x concentrate)

TMB Substrate

Stop Solution

Adhesive Strip (For 96 wells)

Instruction manual

STORAGE

Unopened kit

Opened kit

Store at 2 - 8°C. Do not use the kit beyond the expiration date.

May be stored for up to one month at 2 - 8° C.

Quantity

1(96 wells)

1 x 800 μl

1 x 800 μl

1 x 120μl

1 x 20 ml

2 x 20 ml

1 x 20 ml

1 x 10 ml

1 x 10 ml

4

1

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

3

OTHER SUPPLIES REQUIRED



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

correction wavelength set at 540 nm or 570 nm.

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An incubator which can provide stable incubation conditions up to

37°C±0.5°C.

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Squirt bottle, manifold dispenser, or automated microplate washer.

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Absorbent paper for blotting the microtiter plate.

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100ml and 500ml graduated cylinders.

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Deionized or distilled water.

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Pipettes and pipette tips.

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Test tubes for dilution.

PRECAUTIONS

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

and clothing protection when using this material.

4

SAMPLE COLLECTION AND STORAGE



Serum Use a serum separator tube (SST) and allow samples to clot for

two hours at room temperature or overnight at 4°C before centrifugation

for 15 minutes at 1000 ×g. Remove serum and assay immediately or

aliquot and store samples at -20°C or -80°C. Avoid repeated freeze-thaw

cycles.

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Plasma Collect plasma using citrate, EDTA, or heparin as an

anticoagulant. Centrifuge for 15 minutes at 1000 g within 30 minutes of

collection. Assay immediately or aliquot and store samples at -20° C.

Avoid repeated freeze-thaw cycles.

SAMPLE PREPARATION

Dilute the serum or plasma samples with Sample Diluent(1:2000) before test.

The suggested 2000-fold dilution can be achieved by adding 2μl sample to 98μl

of Sample Diluent. Complete the 2000-fold dilution by adding 6μl of this solution

to 234μl of Sample Diluent.

Note:

1. CUSABIO is only responsible for the kit itself, but not for the samples

consumed during the assay. The user should calculate the possible

amount of the samples used in the whole test. Please reserve sufficient

samples in advance.

2. Samples to be used within 5 days may be stored at 2-8°C, otherwise

samples must be stored at -20°C (≤1month) or -80°C (≤2month) to avoid

loss of bioactivity and contamination.

3. Grossly hemolyzed samples are not suitable for use in this assay.

4. If the samples are not indicated in the manual, a preliminary experiment to

determine the validity of the kit is necessary.

5. Fresh samples without long time storage are recommended for the test.

Otherwise, protein degradation and denaturalization may occur in those

samples and finally lead to wrong results.

5

REAGENT PREPARATION

Note:

 Kindly use graduated containers to prepare the reagent. Please don't

prepare the reagent directly in the Diluent vials provided in the kit.

 Bring all reagents to room temperature (18-25°C) before use for 30min.

 Distilled water is recommended to be used to make the preparation for

reagents. Contaminated water or container for reagent preparation will

influence the detection result.

1. Wash Buffer(1x)- If crystals have formed in the concentrate, warm up to

room temperature and mix gently until the crystals have completely

dissolved. Dilute 20 ml of Wash Buffer Concentrate (25 x) into deionized

or distilled water to prepare 500 ml of Wash Buffer (1 x).

HRP-conjugate (1x) - Centrifuge the vial before opening.

HRP- conjugate requires a 100-fold dilution. A suggested 100-fold dilution

is 10 μl of HRP-conjugate + 990 μl of HRP- conjugate Diluent.

2.

6

ASSAY PROCEDURE

Bring all reagents and samples to room temperature before use. Centrifuge

the sample again after thawing before the assay. It is recommended that all

samples and controls be assayed in duplicate.

1.

2.

Prepare all reagents, and samples as directed in the previous sections.

Refer to the Assay Layout Sheet to determine the number of wells to be

used and put any remaining wells and the desiccant back into the pouch

and seal the ziploc, store unused wells at 4°C.

Set a Blank well without any solution.

Add 100μl of Negative Control, Positive Control or diluted Sample per

well.

Cover with the adhesive strip provided. Incubate for 30 minutes at 37°C.

Aspirate each well and wash, repeating the process two times for a total of

three washes. Wash by filling each well with Wash Buffer (200μl) using a

squirt bottle, multi-channel pipette, manifold dispenser, or autowasher,

and let it stand for 2 minutes, complete removal of liquid at each step is

essential to good performance. After the last wash, remove any remaining

Wash Buffer by aspirating or decanting. Invert the plate and blot it against

clean paper towels.

Add 100μl of HRP-conjugate(1×) to each well (not to Blank!). Cover the

microtiter plate with the adhesive strip. Incubate for 30 minutes at 37°C.

Repeat the aspiration/wash process for five times as in step 6.

Add 90μl of TMB Substrate to each well. Incubate for 20 minutes at 37°C.

Protect from light.

Add 50μl of Stop Solution to each well, gently tap the plate to ensure

thorough mixing.

Take blank well as zero, determine the optical density of each well within

10 minutes, using a microplate reader set to 450 nm.

3.

4.

5.

6.

7.

8.

9.

10.

11.

*Samples may require dilution. Please refer to Sample Preparation section.

7

Note:

1. The final experimental results will be closely related to validity of the

products, operation skills of the end users and the experimental

environments.

2. Samples or reagents addition: Please carefully add samples to wells and

mix gently to avoid foaming. Do not touch the well wall as possible. For

each step in the procedure, total dispensing time for addition of reagents

or samples to the assay plate should not exceed 3 minutes. This will

ensure equal elapsed time for each pipetting step, without interruption.

Duplication of all specimens, although not required, is recommended. To

avoid cross-contamination, change pipette tips between sample additions,

and between reagent additions. Also, use separate reservoirs for each

reagent.

3. Incubation: To ensure accurate results, proper adhesion of plate sealers

during incubation steps is necessary. Do not allow wells to sit uncovered

for extended periods between incubation steps. Once reagents have been

added to the well strips, DO NOT let the strips DRY at any time during the

assay. Incubation time and temperature must be observed.

4. Washing: The wash procedure is critical. Complete removal of liquid at

each step is essential to good performance. After the last wash, remove

any remaining Wash Solution by aspirating or decanting and remove any

drop of water and fingerprint on the bottom of the plate. Insufficient

washing will result in poor precision and falsely elevated absorbance

reading. 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.

5. Controlling of reaction time: Observe the change of color after adding TMB

Substrate (e.g. observation once every 10 minutes), TMB Substrate

should change from colorless or light blue to gradations of blue. If the color

is too deep, add Stop Solution in advance to avoid excessively strong

reaction which will result in inaccurate absorbance reading.

6. TMB Substrate is easily contaminated. TMB Substrate should remain

colorless or light blue until added to the plate. Please protect it from light.

7. Stop Solution should be added to the plate in the same order as the TMB

Substrate. The color developed in the wells will turn from blue to yellow

upon addition of the Stop Solution. Wells that are green in color indicate

that the Stop Solution has not mixed thoroughly with the TMB Substrate.

8

CALCULATION OF RESULTS

For calculation the valence of human IFN-γ antibody, compare the sample well

with control.

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