人免疫球蛋白E(IgE)英文说明书

Human immunoglobulin E（IgE）  人免疫球蛋白E(IgE)英文说明书

FOR RESEARCH USE ONLY

Assay range：7.5μg/ml -240μg/ml 96 determinations

Purpose

This kit allows for the determination of IgE concentrations in Human serum, cell

culture supernates and other biological fluids

Principle of the assay

The kit assay Human IgE level in the sample，use Purified Human IgE antibody to coat

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

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 Human IgE in the samples is then determined by comparing the O.D. of the

samples to the standard curve.

Materials provided with the kit

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

2 HRP-Conjugate reagent 6ml×1 bottle 8

Standard

（480μg/ml） 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

2

membrane

6 Chromogen Solution B 6ml×1 bottle 12 Sealed bags 1

Specimen requirements

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:

240μg/ml 5 Standard 150μl Original density Standard+150μl Standard diluent

120μg/ml 4 Standard 150μl 5 Standard+150μl Standard diluent

60μg/ml 3 Standard 150μl 4 Standard+150μl Standard diluent

30μg/ml 2 Standard 150μl 3 Standard +150μl Standard diluent

15μg/ml 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 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 to each well, evade the

light preservation for 15 min at 37℃

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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 30 min at 37℃.

Add Stopp Solution

Read absorbance at 450nm within 15 min

calculate

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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,

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, 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 and validity

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1．Storage： 2-8℃.

2．validity： six months