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人肿瘤坏死因子α(TNF-α)说明书

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Human TNF-α

 

FOR RESEARCH USE ONLY

 

 

Assay range20 ng/L -400 ng/L 96 DETERMINATIONS

 

Purpose

 

This kit allows for the determination of TNF-α concentrations in Human serum.

 

 

Principle of the assay

 

The kit assay Human TNF-α level in the sample, use Purified Human TNF-α antibody to coat microtiter plate wells, make solid-phase antibody, then add TNF-α to wells, Combined TNF-α antibody which With HRP labeled, become antibody - antigen - enzyme-antibody complex, after washing Compley, Add TMB substrate solution,TMB substrate becomes blue color At HRP enzyme-catalyzed, reaction is terminated by the addition of a sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450 nm. The concentration of Human TNF-α in the samples is then determined by comparing the O.D. of the samples to the standard curve.

 

Materials provided with the kit

 

1

washsolution

20ml×1bottle

7

Stop Solution

6ml×1 bottle

 

 

 

 

 

 

2

HRP-Conjugate reagent

6ml×1 bottle

8

Standard800ng/L

0.5ml×1 bottle

 

 

 

 

 

 

3

Microelisa stripplate

12well×8strips

9

Standard diluent

1.5ml×1bottle

 

 

 

 

 

 

4

Sample diluent

6ml×1 bottle

10

Instruction

1

 

 

 

 

 

 

5

Chromogen Solution A

6ml×1 bottle

11

Closure plate

2

membrane

 

 

 

 

 

6

Chromogen Solution B

6ml×1 bottle

12

Sealed bags

1

 

 

 

 

 

 

 

Specimen requirements

 

  • extract as soon as possible after Specimen collection,and according to the relevant literature, and should be experiment as soon as possible after the extraction. If it can’t, specimen can be kept in -20  to preserve, Avoid repeated freeze-thaw cycles.

 

1

 

 

2. Can’t detect the sample which contain NaN3, because NaN3 inhibits HRP active.

 

Assay procedure

 

1. Dilute and add sample:Dilute Original density Standard as follow table:

 

400ng/L

5

Standard

150µl Original density Standard+150µl Standard diluent

 

 

 

 

 

200ng/L

4

Standard

150µl 5 Standard+150µl Standard diluent

 

 

 

 

 

100ng/L

3

Standard

150µl 4 Standard+150µl Standard diluent

 

 

 

 

 

50ng/L

2

Standard

150µl 3 Standard +150µl Standard diluent

 

 

 

 

 

25ng/L

1

Standard

150µl 2 Standard +150µl Standard diluent

 

 

 

 

 

 

2.add sampleSet blank wells separay (blank comparison wells don’t add sample and HRP-Conjugate reagent, other each step operation is same). testing sample well. add Sample dilution 40µl to testing sample well, then add testing sample 10µl (sample final dilution is 5-fold), add sample to wells , don’t touch the well wall as far as possible, and Gently mix.

 

3.Incubate: After closing plate with Closure plate membrane ,incubate for 30 min at 37. 4.Configurate liquid: 30-fold(or 20-fold) wash solution diluted 30-fold (or 20-fold) with distilled

 

water and reserve.

 

5.washingUncover Closure plate membrane, discard Liquid, dry by swing, add washing buffer to every well, still for 30s then drain, repeat 5 times, dry by pat.

 

6.add enzymeAdd HRP-Conjugate reagent 50µl to each well, except blank well. 7.incubateOperation with 3.

 

8.washingOperation with 5.

 

9.colorAdd Chromogen Solution A 50ul and Chromogen Solution B to each well, evade the light preservation for 10 min at 37

 

10.Stop the reactionAdd Stop Solution50µl to each well, Stop the reaction(the blue color change to yellow color).

 

11.assaytake blank well as zero , Read absorbance at 450nm after Adding Stop Solution and

 

within 15min.

 

Steps description

 

 

 

 

2

 

 

Standard, Sample diluent

 

 

 

 

 

 

 

Add Standard, Sample diluent, incubate for 30 min at 37.

 

 

 

 

 

 

 

Wash 5 time,Add HRP-Conjugate reagent, incubate for 30 min at 37.

 

 

 

 

 

 

 

Wash 5 times,Add Chromogen Solution A and B, incubate for 10 min at 37.

 

 

 

 

 

 

 

Add Stopp Solution

 

 

 

 

 

 

 

Read absorbance at 450nm within 15 min

 

 

 

 

 

 

 

calculate

 

Calculate

 

Take the standard density as the horizontal, the OD value for the vertical ,draw the standard curve on graph paper, Find out the corresponding density according to the sample OD value by the Sample curve, multiplied by the dilution multiple, or calculate the straight line regression equation of the standard curve with the standard density and the OD value ,with the sample OD value in the equation, calculate the sample density, multiplied by the dilution factor,

 

3

 

 

the result is the sample actual density.

 

Important notes

 

  • The kit takes out from the refrigeration environment should be balanced 15-30 minutes in the room temperature, ELISA plates coated if has not use up after opened, the plate should be stored in Sealed bag.

 

  • washing buffer will Crystallization separation, it can be heated the water helps dissolve when dilute . Washing does not affect the result.

 

  • add Sample with sampler Each step, And proofread its accuracy frequently, avoids the experimental error. add sample within 5 min, if the number of sample is much , recommend to use Volley .

 

  • if the testing material content is excessively higher (The sample OD is bigger than the first standard well ),please dilute Sample (n-fold), Please diluente and multiplied by the dilution factor.×n×5.

 

  • Closure plate membrane only limits the disposable use, to avoid cross-contamination.

 

  • The substrate evade the light preservation.

 

  • Please according to use instruction strictly, The test result determination must take the microtiter plate reader as a standard.

 

  • All samples, washing buffer and each kind of reject should according to infective material process.

 

  • Do not mix reagents with those from other lots.

 

 

 

Storage and validity

 

1Storage 2-8.

 

2validity six months

 

 

 

 

 

 

 

 

 

 

 

4

 

Human TNF-α

 

FOR RESEARCH USE ONLY

 

 

Assay range20 ng/L -400 ng/L 96 DETERMINATIONS

 

Purpose

 

This kit allows for the determination of TNF-α concentrations in Human serum.

 

 

Principle of the assay

 

The kit assay Human TNF-α level in the sample, use Purified Human TNF-α antibody to coat microtiter plate wells, make solid-phase antibody, then add TNF-α to wells, Combined TNF-α antibody which With HRP labeled, become antibody - antigen - enzyme-antibody complex, after washing Compley, Add TMB substrate solution,TMB substrate becomes blue color At HRP enzyme-catalyzed, reaction is terminated by the addition of a sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450 nm. The concentration of Human TNF-α in the samples is then determined by comparing the O.D. of the samples to the standard curve.

 

Materials provided with the kit

 

1

washsolution

20ml×1bottle

7

Stop Solution

6ml×1 bottle

 

 

 

 

 

 

2

HRP-Conjugate reagent

6ml×1 bottle

8

Standard800ng/L

0.5ml×1 bottle

 

 

 

 

 

 

3

Microelisa stripplate

12well×8strips

9

Standard diluent

1.5ml×1bottle

 

 

 

 

 

 

4

Sample diluent

6ml×1 bottle

10

Instruction

1

 

 

 

 

 

 

5

Chromogen Solution A

6ml×1 bottle

11

Closure plate

2

membrane

 

 

 

 

 

6

Chromogen Solution B

6ml×1 bottle

12

Sealed bags

1

 

 

 

 

 

 

 

Specimen requirements

 

  • extract as soon as possible after Specimen collection,and according to the relevant literature, and should be experiment as soon as possible after the extraction. If it can’t, specimen can be kept in -20  to preserve, Avoid repeated freeze-thaw cycles.

 

1

 

 

2. Can’t detect the sample which contain NaN3, because NaN3 inhibits HRP active.

 

Assay procedure

 

1. Dilute and add sample:Dilute Original density Standard as follow table:

 

400ng/L

5

Standard

150µl Original density Standard+150µl Standard diluent

 

 

 

 

 

200ng/L

4

Standard

150µl 5 Standard+150µl Standard diluent

 

 

 

 

 

100ng/L

3

Standard

150µl 4 Standard+150µl Standard diluent

 

 

 

 

 

50ng/L

2

Standard

150µl 3 Standard +150µl Standard diluent

 

 

 

 

 

25ng/L

1

Standard

150µl 2 Standard +150µl Standard diluent

 

 

 

 

 

 

2.add sampleSet blank wells separay (blank comparison wells don’t add sample and HRP-Conjugate reagent, other each step operation is same). testing sample well. add Sample dilution 40µl to testing sample well, then add testing sample 10µl (sample final dilution is 5-fold), add sample to wells , don’t touch the well wall as far as possible, and Gently mix.

 

3.Incubate: After closing plate with Closure plate membrane ,incubate for 30 min at 37. 4.Configurate liquid: 30-fold(or 20-fold) wash solution diluted 30-fold (or 20-fold) with distilled

 

water and reserve.

 

5.washingUncover Closure plate membrane, discard Liquid, dry by swing, add washing buffer to every well, still for 30s then drain, repeat 5 times, dry by pat.

 

6.add enzymeAdd HRP-Conjugate reagent 50µl to each well, except blank well. 7.incubateOperation with 3.

 

8.washingOperation with 5.

 

9.colorAdd Chromogen Solution A 50ul and Chromogen Solution B to each well, evade the light preservation for 10 min at 37

 

10.Stop the reactionAdd Stop Solution50µl to each well, Stop the reaction(the blue color change to yellow color).

 

11.assaytake blank well as zero , Read absorbance at 450nm after Adding Stop Solution and

 

within 15min.

 

Steps description

 

 

 

 

2

 

 

Standard, Sample diluent

 

 

 

 

 

 

 

Add Standard, Sample diluent, incubate for 30 min at 37.

 

 

 

 

 

 

 

Wash 5 time,Add HRP-Conjugate reagent, incubate for 30 min at 37.

 

 

 

 

 

 

 

Wash 5 times,Add Chromogen Solution A and B, incubate for 10 min at 37.

 

 

 

 

 

 

 

Add Stopp Solution

 

 

 

 

 

 

 

Read absorbance at 450nm within 15 min

 

 

 

 

 

 

 

calculate

 

Calculate

 

Take the standard density as the horizontal, the OD value for the vertical ,draw the standard curve on graph paper, Find out the corresponding density according to the sample OD value by the Sample curve, multiplied by the dilution multiple, or calculate the straight line regression equation of the standard curve with the standard density and the OD value ,with the sample OD value in the equation, calculate the sample density, multiplied by the dilution factor,

 

3

 

 

the result is the sample actual density.

 

Important notes

 

  • The kit takes out from the refrigeration environment should be balanced 15-30 minutes in the room temperature, ELISA plates coated if has not use up after opened, the plate should be stored in Sealed bag.

 

  • washing buffer will Crystallization separation, it can be heated the water helps dissolve when dilute . Washing does not affect the result.

 

  • add Sample with sampler Each step, And proofread its accuracy frequently, avoids the experimental error. add sample within 5 min, if the number of sample is much , recommend to use Volley .

 

  • if the testing material content is excessively higher (The sample OD is bigger than the first standard well ),please dilute Sample (n-fold), Please diluente and multiplied by the dilution factor.×n×5.

 

  • Closure plate membrane only limits the disposable use, to avoid cross-contamination.

 

  • The substrate evade the light preservation.

 

  • Please according to use instruction strictly, The test result determination must take the microtiter plate reader as a standard.

 

  • All samples, washing buffer and each kind of reject should according to infective material process.

 

  • Do not mix reagents with those from other lots.

 

 

 

Storage and validity

 

1Storage 2-8.

 

2validity six months

 

 

 

 

 

 

Human TNF-α

 

FOR RESEARCH USE ONLY

 

 

Assay range20 ng/L -400 ng/L 96 DETERMINATIONS

 

Purpose

 

This kit allows for the determination of TNF-α concentrations in Human serum.

 

 

Principle of the assay

 

The kit assay Human TNF-α level in the sample, use Purified Human TNF-α antibody to coat microtiter plate wells, make solid-phase antibody, then add TNF-α to wells, Combined TNF-α antibody which With HRP labeled, become antibody - antigen - enzyme-antibody complex, after washing Compley, Add TMB substrate solution,TMB substrate becomes blue color At HRP enzyme-catalyzed, reaction is terminated by the addition of a sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450 nm. The concentration of Human TNF-α in the samples is then determined by comparing the O.D. of the samples to the standard curve.

 

Materials provided with the kit

 

1

washsolution

20ml×1bottle

7

Stop Solution

6ml×1 bottle

 

 

 

 

 

 

2

HRP-Conjugate reagent

6ml×1 bottle

8

Standard800ng/L

0.5ml×1 bottle

 

 

 

 

 

 

3

Microelisa stripplate

12well×8strips

9

Standard diluent

1.5ml×1bottle

 

 

 

 

 

 

4

Sample diluent

6ml×1 bottle

10

Instruction

1

 

 

 

 

 

 

5

Chromogen Solution A

6ml×1 bottle

11

Closure plate

2

membrane

 

 

 

 

 

6

Chromogen Solution B

6ml×1 bottle

12

Sealed bags

1

 

 

 

 

 

 

 

Specimen requirements

 

  • extract as soon as possible after Specimen collection,and according to the relevant literature, and should be experiment as soon as possible after the extraction. If it can’t, specimen can be kept in -20  to preserve, Avoid repeated freeze-thaw cycles.

 

1

 

 

2. Can’t detect the sample which contain NaN3, because NaN3 inhibits HRP active.

 

Assay procedure

 

1. Dilute and add sample:Dilute Original density Standard as follow table:

 

400ng/L

5

Standard

150µl Original density Standard+150µl Standard diluent

 

 

 

 

 

200ng/L

4

Standard

150µl 5 Standard+150µl Standard diluent

 

 

 

 

 

100ng/L

3

Standard

150µl 4 Standard+150µl Standard diluent

 

 

 

 

 

50ng/L

2

Standard

150µl 3 Standard +150µl Standard diluent

 

 

 

 

 

25ng/L

1

Standard

150µl 2 Standard +150µl Standard diluent

 

 

 

 

 

 

2.add sampleSet blank wells separay (blank comparison wells don’t add sample and HRP-Conjugate reagent, other each step operation is same). testing sample well. add Sample dilution 40µl to testing sample well, then add testing sample 10µl (sample final dilution is 5-fold), add sample to wells , don’t touch the well wall as far as possible, and Gently mix.

 

3.Incubate: After closing plate with Closure plate membrane ,incubate for 30 min at 37. 4.Configurate liquid: 30-fold(or 20-fold) wash solution diluted 30-fold (or 20-fold) with distilled

 

water and reserve.

 

5.washingUncover Closure plate membrane, discard Liquid, dry by swing, add washing buffer to every well, still for 30s then drain, repeat 5 times, dry by pat.

 

6.add enzymeAdd HRP-Conjugate reagent 50µl to each well, except blank well. 7.incubateOperation with 3.

 

8.washingOperation with 5.

 

9.colorAdd Chromogen Solution A 50ul and Chromogen Solution B to each well, evade the light preservation for 10 min at 37

 

10.Stop the reactionAdd Stop Solution50µl to each well, Stop the reaction(the blue color change to yellow color).

 

11.assaytake blank well as zero , Read absorbance at 450nm after Adding Stop Solution and

 

within 15min.

 

Steps description

 

 

 

 

2

 

 

Standard, Sample diluent

 

 

 

 

 

 

 

Add Standard, Sample diluent, incubate for 30 min at 37.

 

 

 

 

 

 

 

Wash 5 time,Add HRP-Conjugate reagent, incubate for 30 min at 37.

 

 

 

 

 

 

 

Wash 5 times,Add Chromogen Solution A and B, incubate for 10 min at 37.

 

 

 

 

 

 

 

Add Stopp Solution

 

 

 

 

 

 

 

Read absorbance at 450nm within 15 min

 

 

 

 

 

 

 

calculate

 

Calculate

 

Take the standard density as the horizontal, the OD value for the vertical ,draw the standard curve on graph paper, Find out the corresponding density according to the sample OD value by the Sample curve, multiplied by the dilution multiple, or calculate the straight line regression equation of the standard curve with the standard density and the OD value ,with the sample OD value in the equation, calculate the sample density, multiplied by the dilution factor,

 

3

 

 

the result is the sample actual density.

 

Important notes

 

  • The kit takes out from the refrigeration environment should be balanced 15-30 minutes in the room temperature, ELISA plates coated if has not use up after opened, the plate should be stored in Sealed bag.

 

  • washing buffer will Crystallization separation, it can be heated the water helps dissolve when dilute . Washing does not affect the result.

 

  • add Sample with sampler Each step, And proofread its accuracy frequently, avoids the experimental error. add sample within 5 min, if the number of sample is much , recommend to use Volley .

 

  • if the testing material content is excessively higher (The sample OD is bigger than the first standard well ),please dilute Sample (n-fold), Please diluente and multiplied by the dilution factor.×n×5.

 

  • Closure plate membrane only limits the disposable use, to avoid cross-contamination.

 

  • The substrate evade the light preservation.

 

  • Please according to use instruction strictly, The test result determination must take the microtiter plate reader as a standard.

 

  • All samples, washing buffer and each kind of reject should according to infective material process.

 

  • Do not mix reagents with those from other lots.

 

 

 

Storage and validity

 

1Storage 2-8.

 

2validity six months

 

 

 

 

 

 

Human TNF-α

 

FOR RESEARCH USE ONLY

 

 

Assay range20 ng/L -400 ng/L 96 DETERMINATIONS

 

Purpose

 

This kit allows for the determination of TNF-α concentrations in Human serum.

 

 

Principle of the assay

 

The kit assay Human TNF-α level in the sample, use Purified Human TNF-α antibody to coat microtiter plate wells, make solid-phase antibody, then add TNF-α to wells, Combined TNF-α antibody which With HRP labeled, become antibody - antigen - enzyme-antibody complex, after washing Compley, Add TMB substrate solution,TMB substrate becomes blue color At HRP enzyme-catalyzed, reaction is terminated by the addition of a sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450 nm. The concentration of Human TNF-α in the samples is then determined by comparing the O.D. of the samples to the standard curve.

 

Materials provided with the kit

 

1

washsolution

20ml×1bottle

7

Stop Solution

6ml×1 bottle

 

 

 

 

 

 

2

HRP-Conjugate reagent

6ml×1 bottle

8

Standard800ng/L

0.5ml×1 bottle

 

 

 

 

 

 

3

Microelisa stripplate

12well×8strips

9

Standard diluent

1.5ml×1bottle

 

 

 

 

 

 

4

Sample diluent

6ml×1 bottle

10

Instruction

1

 

 

 

 

 

 

5

Chromogen Solution A

6ml×1 bottle

11

Closure plate

2

membrane

 

 

 

 

 

6

Chromogen Solution B

6ml×1 bottle

12

Sealed bags

1

 

 

 

 

 

 

 

Specimen requirements

 

  • extract as soon as possible after Specimen collection,and according to the relevant literature, and should be experiment as soon as possible after the extraction. If it can’t, specimen can be kept in -20  to preserve, Avoid repeated freeze-thaw cycles.

 

1

 

 

2. Can’t detect the sample which contain NaN3, because NaN3 inhibits HRP active.

 

Assay procedure

 

1. Dilute and add sample:Dilute Original density Standard as follow table:

 

400ng/L

5

Standard

150µl Original density Standard+150µl Standard diluent

 

 

 

 

 

200ng/L

4

Standard

150µl 5 Standard+150µl Standard diluent

 

 

 

 

 

100ng/L

3

Standard

150µl 4 Standard+150µl Standard diluent

 

 

 

 

 

50ng/L

2

Standard

150µl 3 Standard +150µl Standard diluent

 

 

 

 

 

25ng/L

1

Standard

150µl 2 Standard +150µl Standard diluent

 

 

 

 

 

 

2.add sampleSet blank wells separay (blank comparison wells don’t add sample and HRP-Conjugate reagent, other each step operation is same). testing sample well. add Sample dilution 40µl to testing sample well, then add testing sample 10µl (sample final dilution is 5-fold), add sample to wells , don’t touch the well wall as far as possible, and Gently mix.

 

3.Incubate: After closing plate with Closure plate membrane ,incubate for 30 min at 37. 4.Configurate liquid: 30-fold(or 20-fold) wash solution diluted 30-fold (or 20-fold) with distilled

 

water and reserve.

 

5.washingUncover Closure plate membrane, discard Liquid, dry by swing, add washing buffer to every well, still for 30s then drain, repeat 5 times, dry by pat.

 

6.add enzymeAdd HRP-Conjugate reagent 50µl to each well, except blank well. 7.incubateOperation with 3.

 

8.washingOperation with 5.

 

9.colorAdd Chromogen Solution A 50ul and Chromogen Solution B to each well, evade the light preservation for 10 min at 37

 

10.Stop the reactionAdd Stop Solution50µl to each well, Stop the reaction(the blue color change to yellow color).

 

11.assaytake blank well as zero , Read absorbance at 450nm after Adding Stop Solution and

 

within 15min.

 

Steps description

 

 

 

 

2

 

 

Standard, Sample diluent

 

 

 

 

 

 

 

Add Standard, Sample diluent, incubate for 30 min at 37.

 

 

 

 

 

 

 

Wash 5 time,Add HRP-Conjugate reagent, incubate for 30 min at 37.

 

 

 

 

 

 

 

Wash 5 times,Add Chromogen Solution A and B, incubate for 10 min at 37.

 

 

 

 

 

 

 

Add Stopp Solution

 

 

 

 

 

 

 

Read absorbance at 450nm within 15 min

 

 

 

 

 

 

 

calculate

 

Calculate

 

Take the standard density as the horizontal, the OD value for the vertical ,draw the standard curve on graph paper, Find out the corresponding density according to the sample OD value by the Sample curve, multiplied by the dilution multiple, or calculate the straight line regression equation of the standard curve with the standard density and the OD value ,with the sample OD value in the equation, calculate the sample density, multiplied by the dilution factor,

 

3

 

 

the result is the sample actual density.

 

Important notes

 

  • The kit takes out from the refrigeration environment should be balanced 15-30 minutes in the room temperature, ELISA plates coated if has not use up after opened, the plate should be stored in Sealed bag.

 

  • washing buffer will Crystallization separation, it can be heated the water helps dissolve when dilute . Washing does not affect the result.

 

  • add Sample with sampler Each step, And proofread its accuracy frequently, avoids the experimental error. add sample within 5 min, if the number of sample is much , recommend to use Volley .

 

  • if the testing material content is excessively higher (The sample OD is bigger than the first standard well ),please dilute Sample (n-fold), Please diluente and multiplied by the dilution factor.×n×5.

 

  • Closure plate membrane only limits the disposable use, to avoid cross-contamination.

 

  • The substrate evade the light preservation.

 

  • Please according to use instruction strictly, The test result determination must take the microtiter plate reader as a standard.

 

  • All samples, washing buffer and each kind of reject should according to infective material process.

 

  • Do not mix reagents with those from other lots.

 

 

 

Storage and validity

 

1Storage 2-8.

 

2validity six

 
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