小鼠基質金屬蛋白酶9（MMP-9）Elisa試劑盒說明書

【資料簡介】

小鼠基質金屬蛋白酶9（MMP-9）Elisa試劑盒說明書

本試劑盒僅供研究使用。

檢測范圍： 96T

1.5μg/L -65μg/L

使用目的：

本試劑盒用于測定小鼠血清、血漿及相關液體樣本中基質金屬蛋白酶-9（MMP-9）含量。

實驗原理

小鼠基質金屬蛋白酶9（MMP-9）Elisa試劑盒說明書本試劑盒應用雙抗體夾心法測定標本中小鼠基質金屬蛋白酶-9（MMP-9）水平。用純化

的小鼠基質金屬蛋白酶-9（MMP-9）抗體包被微孔板，制成固相抗體，往包被單抗的微孔中

依次加入基質金屬蛋白酶-9（MMP-9），再與HRP 標記的基質金屬蛋白酶-9（MMP-9）抗體

結合，形成抗體-抗原-酶標抗體復合物，經過*洗滌后加底物TMB 顯色。TMB 在HRP

酶的催化下轉化成藍色，并在酸的作用下轉化成zui終的黃色。顏色的深淺和樣品中的基質金

屬蛋白酶-9（MMP-9）呈正相關。用酶標儀在450nm 波長下測定吸光度（OD 值），通過標

準曲線計算樣品中小鼠基質金屬蛋白酶-9（MMP-9）濃度。

試劑盒組成

1 30 倍濃縮洗滌液 20ml×1 瓶 7 終止液 6ml×1 瓶

2 酶標試劑 6ml×1 瓶 8 標準品（120μg/L） 0.5ml×1 瓶

3 酶標包被板 12 孔×8 條 9 標準品稀釋液 1.5ml×1 瓶

4 樣品稀釋液 6ml×1 瓶 10 說明書 1 份

5 顯色劑A 液 6ml×1 瓶 11 封板膜 2 張

6 顯色劑B 液 6ml×1/瓶 12 密封袋 1 個

標本要求

1．標本采集后盡早進行提取，提取按相關文獻進行，提取后應盡快進行實驗。若不能

馬上進行試驗，可將標本放于-20℃保存，但應避免反復凍融

2．不能檢測含NaN3 的樣品，因NaN3 抑制辣根過氧化物酶的（HRP）活性。

操作步驟

1. 標準品的稀釋：本試劑盒提供原倍標準品一支，用戶可按照下列圖表在小試管中進行稀

釋。

60μg/L 5 號標準品 150μl 的原倍標準品加入150μl 標準品稀釋液

30μg/L 4 號標準品 150μl 的5 號標準品加入150μl 標準品稀釋液

15μg/L 3 號標準品 150μl 的4 號標準品加入150μl 標準品稀釋液

7.5μg/L 2 號標準品 150μl 的3 號標準品加入150μl 標準品稀釋液

3.75μg/L 1 號標準品 150μl 的2 號標準品加入150μl 標準品稀釋液

2. 加樣：分別設空白孔（空白對照孔不加樣品及酶標試劑，其余各步操作相同）、標準孔、

待測樣品孔。在酶標包被板上標準品準確加樣50μl，待測樣品孔中先加樣品稀釋液40μl，

然后再加待測樣品10μl（樣品zui終稀釋度為5 倍）。加樣將樣品加于酶標板孔底部，盡

量不觸及孔壁，輕輕晃動混勻。

3. 溫育：用封板膜封板后置37℃溫育30 分鐘。

4. 配液：將30 倍濃縮洗滌液用蒸餾水30 倍稀釋后備用

5. 洗滌：小心揭掉封板膜，棄去液體，甩干，每孔加滿洗滌液，靜置30 秒后棄去，如此

重復5 次，拍干。

6. 加酶：每孔加入酶標試劑50μl，空白孔除外。

7. 溫育：操作同3。

8. 洗滌：操作同5。

9. 顯色：每孔先加入顯色劑A50μl，再加入顯色劑B50μl，輕輕震蕩混勻，37℃避光顯色

15 分鐘.

10. 終止：每孔加終止液50μl，終止反應（此時藍色立轉黃色）。

11. 測定：以空白空調零，450nm 波長依序測量各孔的吸光度（OD 值）。 測定應在加終止

液后15 分鐘以內進行。

操作程序總結：

計算

以標準物的濃度為橫坐標，OD 值為縱坐標，在坐標紙上繪出標準曲線，根據樣品的

OD 值由標準曲線查出相應的濃度；再乘以稀釋倍數；或用標準物的濃度與OD 值計算出標

準曲線的直線回歸方程式，將樣品的OD 值代入方程式，計算出樣品濃度，再乘以稀釋倍數，

即為樣品的實際濃度。

注意事項

1．試劑盒從冷藏環境中取出應在室溫平衡15-30 分鐘后方可使用，酶標包被板開封后如未

用完，板條應裝入密封袋中保存。

2．濃洗滌液可能會有結晶析出，稀釋時可在水浴中加溫助溶，洗滌時不影響結果。

3．各步加樣均應使用加樣器，并經常校對其準確性，以避免試驗誤差。一次加樣時間

控制在5 分鐘內，如標本數量多，推薦使用排槍加樣。

4． 請每次測定的同時做標準曲線，做復孔。如標本中待測物質含量過高（樣本OD 值

大于標準品孔*孔的OD 值），請先用樣品稀釋液稀釋一定倍數（n 倍）后再測定，計

算時請zui后乘以總稀釋倍數（×n×5）。

5． 封板膜只限一次性使用，以避免交叉污染。

6．底物請避光保存。

7．嚴格按照說明書的操作進行，試驗結果判定必須以酶標儀讀數為準.

8．所有樣品，洗滌液和各種廢棄物都應按傳染物處理。

9．本試劑不同批號組分不得混用。

10. 如與英文說明書有異，以英文說明書為準。

保存條件及有效期

1．試劑盒保存：；2-8℃。

2．有效期：6 個月

Human MMP-9

FOR RESEARCH USE ONLY

Assay range：45μg/L -1200μg/L 96 determinations

Purpose

This kit allows for the determination of MMP-9 concentrations in Human serum, cell

culture supernates and other biological fluids

Principle of the assay

The kit assay Human MMP-9 level in the sample，use Purified Human 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 goat anti-Human 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 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 Stopp Solution 6ml×1 bottle

2 HRP-Conjugate reagent 6ml×1 bottle 8

Standard

（2400μg/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

Specimen requirements

RD

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:

1200μg/L 5 Standard 150μl Original density Standard+150μl Standard diluent

600μg/L 4 Standard 150μl 5 Standard+150μl Standard diluent

300μg/L 3 Standard 150μl 4 Standard+150μl Standard diluent

150μg/L 2 Standard 150μl 3 Standard +150μl Standard diluent

75μg/L 1 Standard 150μl 2 Standard +150μl Standard diluent

2.add sample：Set 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.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℃

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

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

1．Storage： 2-8℃.

2．validity： six months

小鼠基質金屬蛋白酶9（MMP-9）Elisa試劑盒說明書