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Rat Matrix metalloproteinase-9 (MMP-9)
FOR RESEARCH USE ONLY
Assay range ： 2µg/L -80µg/L 96 determinations
Purpose
This kit allows for the determination of MMP-9 concentrations in Rat serum.
Principle of the assay
T he kit assay Rat MMP-9 level in the sample, use Purified Rat MMP-9 antibody to coat
microtiter plate wells, make solid-phase antibody, then add MMP-9 to wells, Combined MMP-9
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 Rat MMP-9 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（160µg/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
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.
IBL
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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:
80µg/L
5 Standard 150µl Original density Standard +150µl Standard diluent
40µg/L
4 Standard 150µl 5 Standard +150µl Standard diluent
20µg/L
3 Standard 150µl 4 Standard +150µl Standard diluent
10µg/L
2 Standard 150µl 3  Standard +150µl Standard diluent
5µg/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 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
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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 Stop 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,
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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
1．Storage： 2-8℃.
2．validity： six months