Human serotonin (5-HT) enzyme-linked immunoassay kit instructions - Database & Sql Blog Articles

people

5

Serotonin

(5-HT)

Enzyme-linked immunosorbent assay

ELISA

) Kit Instruction Manual

This reagent is for research use only

Purpose: This kit is used to determine human serum, cell supernatant and related liquid samples.

5

Serotonin

(5-HT)

The content.

Experimental principle:

This kit uses the double antibody sandwich method to determine the person in the specimen.

5

Serotonin

(5-HT)

Level. Purified person

5

Serotonin

(5-HT)

The antibody is coated with a microplate to prepare a solid phase antibody, which is sequentially added to the microwell of the coated monoclonal antibody.

5

Serotonin

(5-HT)

And then

HRP

marked

5

Serotonin

(5-HT)

Antibody binding

-

antigen

-

Enzyme-labeled antibody complex, after thorough washing, adding substrate

TMB

Color development.

TMB

in

HRP

The enzyme is converted to blue by catalysis and converted to the final yellow color by the action of an acid. The depth of the color and the person in the sample

5

Serotonin

(5-HT)

Positive correlation. Using a microplate reader

450nm

Absorbance at wavelength (

OD

Value), calculate the person in the sample through the standard curve

5

Serotonin

(5-HT)

concentration.

Kit composition

:

Kit composition

48

Hole configuration

96

Hole configuration

save

Instruction manual

1

Share

1

Share

Sealing film

2

sheet(

48

)

2

sheet(

96

)

sealed bag

1

One

1

One

Enzyme label coated plate

1

×

48

1

×

96

2-8

°C save

Standard:

900ng/ml

0.5ml

×

1

bottle

0.5ml

×

1

bottle

2-8

°C save

Standard dilution

1.5ml

×

1

bottle

1.5ml

×

1

bottle

2-8

°C save

Enzyme standard reagent

3 ml

×

1

bottle

6 ml

×

1

bottle

2-8

°C save

Sample diluent

3 ml

×

1

bottle

6 ml

×

1

bottle

2-8

°C save

Reagent

A

liquid

3 ml

×

1

bottle

6 ml

×

1

bottle

2-8

°C save

Reagent

B

liquid

3 ml

×

1

bottle

6 ml

×

1

bottle

2-8

°C save

Stop solution

3ml

×

1

bottle

6ml

×

1

bottle

2-8

°C save

Concentrated washing solution

(

20ml

×

20

Times)×

1

bottle

(

20ml

×

30

Times)×

1

bottle

2-8

°C save

Sample processing and requirements

:

1.

Serum: room temperature blood naturally coagulates

10-20

Minute, centrifuge

20

Minutes or so

2000-3000

turn

/

Minute). The supernatant is carefully collected, and if precipitation occurs during storage, it should be centrifuged again.

2.

Plasma: should be selected according to the requirements of the specimen

EDTA

Or sodium citrate as an anticoagulant, mixed

10-20

After a minute, centrifuge

20

Minutes or so

2000-3000

turn

/

Minute). The supernatant is carefully collected, and if a precipitate forms during storage, it should be centrifuged again.

3.

Urine: Collect with sterile tube, centrifuge

20

Minutes or so

2000-3000

turn

/

Minute). The supernatant is carefully collected, and if a precipitate forms during storage, it should be centrifuged again. The chest and ascites and cerebrospinal fluid are referred to.

4.

Cell culture supernatant: When detecting secreted components, collect them in a sterile tube. Centrifugation

20

Minutes or so

2000-3000

turn

/

Minute). Collect the supernatant carefully. When detecting intracellular components, use

PBS

(

PH7.2-7.4

Dilute the cell suspension and reach the cell concentration

100

Ten thousand

/ml

about. By repeated freezing and thawing, the cells are destroyed and the intracellular components are released. Centrifugation

20

Minutes or so

2000-3000

turn

/

Minute). Collect the supernatant carefully. If a precipitate forms during storage, it should be centrifuged again.

5.

Tissue specimen: After cutting the specimen, weigh the weight. Add a certain amount

PBS

,

PH7.4

. It was quickly frozen and stored in liquid nitrogen for use. Specimen remains after melting

2-8

°C temperature. Add a certain amount

PBS

(

PH7.4

), the specimen is homogenized by hand or homogenizer. Centrifugation

20

Minutes or so

2000-3000

turn

/

Minute). Collect the supernatant carefully. One part of the package is to be tested, and the rest is frozen for use.

6.

The specimens should be extracted as soon as possible after collection, and the extraction should be carried out according to the relevant literature. The experiment should be carried out as soon as possible after extraction. If the test cannot be performed immediately,

Put the specimen in

-20

°C save, but

Should avoid repeated freezing and thawing

.

7.

Cannot detect samples containing NaN3

,because

NaN3

Inhibition of horseradish peroxidase

HRP

)active.

Steps

1.

Standard dilution and loading: standard wells on the enzyme label coating

10

Hole, add standard in the first and second holes respectively

100μl

And then add standard dilutions to the first and second wells.

50μl

, mix; then take each from the first hole and the second hole

100μl

Add to the third hole and the fourth hole respectively, and then add the standard dilution solution to the third and fourth holes respectively.

50μl

, mix; then take each of the third and fourth holes

50μl

Discard, take each

50μl

Add to the fifth and sixth holes, respectively, and add standard dilutions to the fifth and sixth holes.

50ul

, mix; after mixing, take each from the fifth and sixth holes

50μl

Add to the seventh and eighth holes, respectively, and add standard dilutions to the seventh and eighth holes.

50μl

, after mixing, take the seventh and eighth holes respectively

50μl

Add to the ninth and tenth holes, and add the standard dilution separately in the ninth and tenth holes.

50μl

, after mixing, take each from the ninth and tenth holes

50μl

Discard. (The amount of each well after dilution is

50μl

, the concentration is

600ng/ml

,

400ng/ml

,

200ng/ml

,

100ng/ml

,

50ng/ml

).

2.

Adding samples: Set blank holes separately (the blank control holes are not added with the sample and the enzyme standard reagent, the other steps are the same), and the sample holes to be tested. Add the sample diluent to the sample well to be tested on the enzyme label coated plate.

40μl

And then add the sample to be tested

10μl

(The final dilution of the sample is

5

Double). Add the sample to the bottom of the well of the microplate, try not to touch the wall of the well, and shake gently to mix.

3.

Incubation: sealing with a sealing film

37

°C incubation

3

0

minute.

4.

Dosing: will

30

(

48T

of

20

Double) concentrated washing solution with distilled water

30

(

48T

of

20

Double) diluted and ready for use.

5.

Washing: Carefully remove the sealing film, discard the liquid, dry it, fill each well with the washing solution, and let it stand.

30

Discard after a second, repeat

5

Times, shoot dry.

6.

Add enzyme: add enzyme label reagent to each well

50μl

Except for blank holes.

7.

Incubation: operation is the same

3

.

8.

Washing: operation is the same

5

.

9.

Color development: first add coloring agent to each well

A50μl

, then add the developer

B50μl

, gently shake and mix,

37

°C dark color

15

minute

.

10.

Termination: add stop solution per well

50μl

, terminate the reaction (the blue color turns yellow at this time).

11.

Determination: with blank air conditioner zero,

450nm

The wavelength measures the absorbance of each well sequentially (

OD

value). The determination should be after adding the stop solution

15

Within minutes.

Precautions:

1.

The kit should be removed from the refrigerated environment and should be balanced at room temperature.

15-30

It can be used after a minute, and if the enzyme label is unsealed after being opened, the slats should be stored in a sealed bag.

2.

Concentrated washing liquid may crystallize out. When diluted, it can be heated and dissolved in a water bath. The washing will not affect the result.

3.

The sampler should be used for each step, and the accuracy should be corrected frequently to avoid test errors. It is best to control the loading time once.

5

Within minutes, if there are a large number of specimens, it is recommended to use a gun to load.

4.

Please make a standard curve at the same time of each measurement, it is best to make a double hole. If the content of the substance to be tested in the specimen is too high (sample

OD

The value is greater than the first hole of the standard hole

OD

Value), please dilute a certain multiple with the sample diluent (

n

Multiply and then measure. When calculating, please multiply by the total dilution factor (×

n

×

5

).

5.

The sealing film is intended for single use only to avoid cross-contamination.

6.

Please keep the substrate away from light.

7.

Strictly follow the instructions of the manual, the test results must be based on the microplate reader reading

.

8.

All samples, washings and various wastes should be treated as infectious materials.

9.

The different batch components of this reagent must not be mixed.

10.

If it is different from the English manual, the English manual shall prevail.

Calculation:

Taking the concentration of the standard as the abscissa,

OD

The value is the ordinate,

Draw a standard curve on the coordinate paper, depending on the sample

OD

The value is determined from the standard curve by the corresponding concentration; multiplied by the dilution

Multiples; or the concentration of the standard

OD

Value calculation

Quasi-curve linear regression equation, the sample

OD

value

Substituting the equation, calculating the sample concentration, multiplying by dilution

The multiple is the actual concentration of the sample.

(This picture is for reference only)

Kit performance:

1.

Sample linear regression and expected concentration correlation coefficient

R

Value

0.92

the above.

2.

Within and within the batch should be less than

9%

with

15%

examination range:

15ng/ml-750ng/ml

Storage conditions and expiration date:

1.

Kit storage:

2-8

°C

.

2

. Validity period:

6

Month