Rat Interleukin 1β（IL-1β）ELISA Kit

Rat Interleukin 1β（IL-1β）ELISA Kit

FOR RESEARCH USE ONLY

INTRODUCTION

Assay range：1 ng/L -40 ng/L 96 determinations

Purpose

This kit allows for the determination of IL-1 β concentrations in Rat

serum, cell culture supernates and other biological fluids

PRINCIPLE OF TEST

The kit assay Rat IL-1 β level in the sample ， use Purified Rat IL-1 β

antibody to coat microtiter plate wells, make solid-phase antibody, then add

IL-1 β to wells, Combined IL-1 β antibody which With HRP labeled,become

antibody - antigen - enzyme-antibody complex, after washing Completely, 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 IL-1 β in the samples is then

determined by comparing the O.D. of the samples to the standard curve.

COMPOSITION OF THE KIT

1 wash solution 20ml×1bottle 7 Stop Solution 6ml×1 bottle

2 HRP-Conjugate

reagent 6ml×1 bottle 8

Standard

（80ng/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

membrane 2

6 Chromogen Solution

B 6ml×1 bottle 12 Sealed bags 1

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SAMPLE PREPARATION

1. 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.

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:

40 ng/L 5 Standard 150μl Original density Standard+150μl Standard diluent

20 ng/L 4 Standard 150μl 5 Standard+150μl Standard diluent

10 ng/L 3 Standard 150μl 4 Standard+150μl Standard diluent

5 ng/L 2 Standard 150μl 3 Standard +150μl Standard diluent

2.5 ng/L 1 Standard 150μl 2 Standard +150μl Standard diluent

2.add sample：Set blank wells separately (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.washing：Uncover Closure plate membrane, discard Liquid, dry by swing,

add washing buffer to every well, still for 30s then drain, repeat 5 times, dry by

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pat.

6.add enzyme：Add HRP-Conjugate reagent 50μl to each well, except blank

well.

7.incubate：Operation with 3.

8.washing：Operation with 5.

9.color：Add Chromogen Solution A 50ul and Chromogen Solution B 50ul to

each well, evade the light preservation for 10 min at 37℃

10.Stop the reaction：Add Stop Solution50μl to each well, Stop the reaction(the

blue color change to yellow color).

11.assay：take blank well as zero , Read absorbance at 450nm after Adding

Stop Solution and within 15min.

Steps description

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℃.

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Add Stop Solution

Read absorbance at 450nm within 15 min

calculate

CALCULATION OF RESULT

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, the result is the sample actual density.

IMPORTANT NOTES

1. 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.

2. washing buffer will Crystallization separation, it can be heated the water

helps dissolve when dilute . Washing does not affect the result.

3. add Sample with sampler Each step, And proofread its accuracy frequently,

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avoids the experimental error. add sample within 5 min, if the number of

sample is much , recommend to use Volley .

4. 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）.

5. Closure plate membrane only limits the disposable use, to avoid

cross-contamination.

6. The substrate evade the light preservation.

7. Please according to use instruction strictly, The test result determination

must take the microtiter plate reader as a standard.

8. All samples, washing buffer and each kind of reject should according to

infective material process.

9. Do not mix reagents with those from other lots.

STORAGE CONDITIONS

? The unopened kit shall be stored at [2-8 ℃] .

? For opened kit can be stored at [2-8 ℃] for up to 1 month. If not be used

recently, the standard should be kept in -20 ℃.

? validity： six months