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Rat Integrins（ITG）ELISA kit
FOR RESEARCH USE ONLY
Assay range： 1.5ng/L -70ng/L 96 determinations
Purpose
This kit allows for the determination of ITG concentrations in Rat serum, cell culture
supernates and other biological fluids
Principle of the assay
The kit assay Rat ITG level in the sample，use Purified Rat ITG antibody to coat
microtiter plate wells, make solid-phase antibody, then add ITG to wells, Combined ITG
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 Rat ITG 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 Stop Solution 6ml×1 bottle
2 HRP-Conjugate reagent 6ml×1 bottle 8 Standard （120ng/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
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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
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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.
大鼠整合素（ITG）ELISA試劑盒技術(shù)參數(shù) 2. Can’t detect the sample which contain NaN3, becauseNaN3 inhibits HRP active.
Assay procedure
1. Dilute and add sample:DiluteOriginal densityStandard as follow table:
60ng/L
5Standard150µlOriginal densityStandard+150µlStandard diluent
30ng/L
4Standard150µl5 Standard+150µlStandard diluent
15ng/L
3Standard150µl 4Standard+150µlStandard diluent
7.5ng/L
2Standard150µl 3 Standard+150µlStandard diluent
3.75ng/L
1Standard150µl 2 Standard+150µlStandard diluent
2.add sample：Set blank wells separay （blank comparison wells don’t add sample and
HRP-Conjugate reagent, other each step operation issame）. 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 wellwall 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, dryby 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, exceptblank 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.
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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℃.
Add Stopp Solution
Read absorbance at 450nm within 15 min
calculate
Calculate
Take the standard density as the horizontal, the ODvalue 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
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sample OD value in the equation, calculate the sample density, multiplied by the dilution factor,
the result is the sample actual density.
大鼠整合素（ITG）ELISA試劑盒技術(shù)參數(shù) 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 thenumber 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 rejectshould according to infective material
process.
9. Do not mix reagents with those from other lots. Storage and validity
1．Storage：2-8℃.
2．validity：six months
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