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人的金屬蛋白酶 9 定量分析酶聯免疫檢測試劑盒

本試劑盒僅供科研使用 。用於體外定量檢測人血清 、血漿或細胞培養上清液中的 MMP-9 濃度 。使用前請仔細閱讀說明書並檢查試劑組

分是否完整 。如有產品包裝破損或質量投拆 ，請在收到貨一個月之內聯係尊龍凱時 。

MMP-9簡介 ：

MMPs蛋白是一組具有鋅指結構的鈣依賴的能夠降解胞間基質成分的一組蛋白的統稱 。MMP-9是該蛋白家族成員中的研究較多

的一種 ，是一種明膠酶（明膠酶B） 。MMP-9有許多作用底物 ，如Ⅳ 、Ⅴ、Ⅶ 、Ⅹ 、Ⅺ型膠原 、蛋白聚糖的核心蛋白 、明膠 、纖維

粘連蛋白 、層粘連蛋白 、彈性蛋白等 。

結構上 ，MMP-9的催化區包括3個重複的型纖維連接蛋白結構域 ，與明膠或彈性蛋白有高度的親和力 。MMP-9還有脯氨酸豐富的

結構域和含鋅指結構的結構域 ，MMP-9包含一個V型的膠原蛋白結構域,這個結構域有高度的糖基化作用 ，,它影響底物的特異性以

及有抗衰變的作用 。

MMP-9是以酶原的形式從胞內分泌到胞外 ，可被MMP-3 、MMP-2或者次氯酸裂解 ， MMP-3可能是MMP-9最有效的激活劑 。金屬蛋白酶

組織抑製因子1（TIMP-1）作為MMP-9的抑製劑 ，與MMP-9的酶原或活化後酶的催化區的羧末端特異性結合 ，形成複合物。

MMP-9可由角化細胞 、單核細胞 、巨噬細胞和多形核白細胞等產生 ，在一些炎性反應的過程中常伴隨著MMP-9的產生 。

檢測原理:

本試劑盒采用雙抗體夾心ELISA法檢測樣本中MMP-9 的濃度 。MMP9 捕獲抗體已預包被於酶標板上 ，當加入標本或參考品時 ，其中的

MMP-9 會與捕獲抗體結合 ，其它遊離的成分通過洗滌的過程被除去 。當加入生物素化的抗人MMP-9 抗體後 ，抗人MMP-9 抗體與MMP-9 接合 ，

形成夾心的免疫複合物 ，其它遊離的成分通過洗滌的過程被除去 。隨後加入辣根過氧化物酶標記的親合素 。生物素與親合素特異性結合 ，

親合素連接的酶就會與夾心的免疫複合物連接起來 ；其它遊離的成分通過洗滌的過程被除去 。最後加入顯色劑 ，若樣本中存在MMP-9 將會

形成免疫複合物 ，辣根過氧化物酶會催化無色的顯色劑氧化成藍色物質 ，在加入終止液後呈黃色 。通過酶標儀檢測 ，讀其450nm處的OD值 ，

MMP-9 濃度與OD450值之間呈正比 ，通過參考品繪製標準曲線 ，對照未知樣本中OD值 ，即可算出標本中MMP-9 濃度 。

人MMP-9定量分析酶聯免疫檢測試劑盒組成:

組分 規格（96T/48T）

人MMP-9預包被板 12條/6條

5X標準品稀釋液 10ml/5ml

人MMP-9標準品 2/1支(凍幹)

人MMP-9生物素化抗體 10ml/5ml

親和素連接的HRP酶 10ml/5ml

濃縮洗滌液 20× 30ml/15ml

TMB底物 10ml/5 ml

中止液 5ml/3 ml

封板膠紙 3/2張

說明書 1份

標本收集:

1.標本的收集請按下列流程進行操作 ；

A.細胞上清標本離心去除懸浮物後即可 ；

B.血清標本應是自然凝固後 ，取上清 ，避免在冰箱中凝固血液 ；

C.血漿標本 ，推薦用EDTA的方法收集 ；

D.標本收集後請分裝 ，若待測樣本不能及時檢測 ，凍存於－20℃ ，避免反複凍融 。

2.血清標本不應添加任何防腐劑或抗凝劑 ；

3.標本應清澈透明 ，檢測前樣本中如有懸浮物應通過離心去除 。

4.請勿使用溶血高血脂或汙染的標本檢測 ，否則結果將不準確 。注 ：人血清或血漿樣本請用標準品稀釋液稀釋後再檢測 。

注意事項:

1.試劑盒請保存在2～8℃ 。

2.濃縮洗滌液因在低溫下可能有結晶 ，請水浴加熱使結晶完全溶解後再配製工作液 。

3.標準品複溶加樣後 ，剩餘部份請丟棄 。

4.底物請勿接觸氧化劑和金屬 。

5.加樣時 ，請及時更換槍頭 ，避免交叉汙染 。

6.嚴禁混用不同批號的試劑盒組份 。

7.充分混勻對保證反應結果的準性很重要 ，在加液後請輕輕叩擊邊緣以保證混勻 。

8.室溫反應 ，請嚴格控製在25～28℃ 。

9.洗滌過程是至關重要的 ，洗滌不充分會使精確度下降並導致結果誤差較大 。

10.試驗中標準品和樣本檢測時建議作雙複孔 。

11.加樣過程中避免氣泡的產生 。

12.血清和血漿標本的檢測時 ，檢測抗體的孵育時間應適當延長 。

檢測前準備工作:

1.試劑盒自冰箱中取出後應置室溫（25～28℃）平衡20分鍾 ；每次檢測後剩餘試劑請及時於2～8℃保存 。

2.將濃縮洗滌液用雙蒸水或去離子水稀釋(1份加19份水) 。

3. 將5×標準品稀釋液用雙蒸水或去離子水稀釋(1份加4份水) 。

4.標準品: 按標簽複溶體積加入1X標準品稀釋液複溶使MMP-9終濃度達到2000pg/ml ，室溫反應 ，請嚴格控製在25～28℃ ，靜置15~20分鍾後

輕輕混懸（建議抽吸幾次）待徹底溶解 ，用標準品稀釋液倍比梯度稀釋後依次加入檢測孔中 。（標準曲線取七個點 ，最高濃度為2000 pg/ml,

標準品稀釋液直接加入作為0濃度.）

洗滌方法:

自動洗板機或人工洗板 ：每孔洗滌液為300ul ，注入與吸出間隔15-30秒 。洗板5次 。最後一次洗板完成後將板倒扣著在厚吸水紙上用力拍幹。

實驗過程需自備的材料 ：

1.不同規格的加樣槍及相應的槍頭 ；

2.酶標儀；

3.自動洗板機 ；

4.去離子水或雙蒸水 ；

操作步驟:

1.通過計算並確定一次性實驗所需的板條數 ，取出所需板條放置在框架內 ，暫時用不到板條請放回鋁箔袋密封 ，保存於4℃ 。

2.建議設置本底較正孔 ，即空白孔,設置方法為該孔隻加TMB顯色液和中止液 。每次實驗均需做標準品對照並畫出標準曲線 。

3.分別將標本或不同濃度標準品(100ul/孔)加入相應孔中 ，用封板膠紙封住反應孔 ，室溫（25～28℃）孵育 120 分鍾 。如果是血清血漿樣

本 ，不同樣本稀釋比例不一樣 ，一般範圍在 400~1500 倍 ，如無明確範圍 ，建議從 400 倍稀釋 ，如果樣本濃度過高 ，超過檢測範圍 ，請加大

稀釋倍數後重新稀釋檢測 。

4.洗板5次 ，且最後一次置厚吸水紙上拍幹 。

5.加入生物素化抗體工作液(100ul/孔) 。用封板膠紙封住反應孔 ，室溫（25～28℃）孵育60分鍾 。

6.洗板5次 ，且最後一次置厚吸水紙上拍幹 。

7.加入親和素連接的HRP酶工作液(100ul/孔) 。用封板膠紙封住反應孔 ，避光室溫（25～28℃）孵育20分鍾 。

上海尊龍凱時生物科技有限公司 www.ruianjituan.com人MMP-9標準曲線

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0 62.5 125 250 500 1000 2000

人MMP-9濃度（pg/ml）

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D值

上海尊龍凱時生物科技有限公司 www.ruianjituan.com

8.洗板5次 ，且最後一次置厚吸水紙上拍幹 。

9.加入顯色劑TMB100ul/孔 ，避光室溫（25～28℃）孵育20分鍾 。

10.加入終止液50ul/孔,混勻後即刻測量OD450值 。

結果判斷:

1.複孔的值在20%的差異範圍內結果才有效 ，複孔的值平均後可作為測量值 。

2.每個標準品或標本的OD值應減去本底校正孔的OD值 。

3.手工繪製標準曲線 。以標準品濃度作橫坐標 ，OD值作縱坐標 ，以平滑線連接各標準品的坐標點 。通過標本的OD值可在標準曲線上查出其

濃度 。

4.若標本 OD 值高於標準曲線上限 ，應適當稀釋後重測 ，計算濃度時應乘以稀釋倍數 。

典型數值和參考曲線

濃度pg/ml 典型OD值1 典型OD值2 OD平均值

0 0.1273 0.1547 0.141

62.5 0.2852 0.295 0.2901

125 0.3438 0.3632 0.3535

250 0.5779 0.5966 0.58725

500 0.8491 0.8733 0.8612

1000 1.3001 1.3866 1.34335

2000 1.9914 2.304 2.1477

人MMP-9參考標準曲線

注意 ：本圖僅供參考 ，應以同次試驗標準品所繪標準曲線計算標本含量 。

靈敏度 ，特異性和重複性:

1.靈敏度 ：多次重複結果表明 ，最小檢出量為7.3pg/ml 。

2.特異性 ：不與MMP-1 、MMP-2 、MMP-3 、MMP-7 、MMP-8 、MMP-10 、MMP-13 、TIMP-2 、TIMP-3 、TIMP-4等反應 。

3.重複性 ：板內 ，板間變異係數均<10%.

參考文獻:

1. Tamura, T. et al. (1996) Endocrinology 137:3729.

2. Okamoto, T. et al. (1997) J. Biol. Chem. 272:6059.

3. Cullen, B. et al. (1997) Int. J. Biochem. Cell Biol. 29:241.

4. Maeda, A. and R.A. Sobel (1996) J. Neuropathol. Exp. Neurol. 55:300.

5. Ueda, Y. et al. (1996) Am. J. Pathol. 148:611.

6. Ito, A. et al. (1996) J. Biol. Chem. 271:14657.上海尊龍凱時生物科技有限公司 www.ruianjituan.com

ELISA Kit for the Quantitative Analysis of Human MMP-9

The human MMP-9 ELISA (enzyme-linked immunosorbent assay) kit is used for detection of human MMP-9 in cell culture

supernatants,human serum and plasma.THE ELISA KIT IS FOR RESEARCH USE ONLY. Please read this instruction manual carefully

and check out the material provided before use, and you can contact with our company if any questions. You can enter our website or call

us for other aim.

Introduction

Matrix metalloproteinases are a family of zinc and calcium dependent endopeptidases with the combined ability to degrade all the

components of the extracellular matrix. MMP 9 (gelatinase B) can degrade a broad range of substrates including gelatin, collagen types

Ⅳ ，Ⅴ ，Ⅶ ，Ⅹand Ⅺ, fibronectin , laminin ,elastin and proteoglycan core protein.

Structurally, MMP9 have a gelatinbinding domain consisting of three contiguous fibronectin type II units, which have high appetency

with gelatin and elastin . MMP9 have a catalytic domain containing the zinc binding site, a proline rich linker region, and a gelatin V

domain with high glycosylation which can select different substrate and resist degradation .

The proenzyme of MMP-9 is secreted into extracellular matrix first, and cleaved into matured MMP-9 by MMP-3,MMP-2 or

hypochlorous acid . It is believed that MMP-3 is the most effective activator to MMP-9 . The interstitial collagenase (MMP1) as a inhibitor

can combine the carboxyl terminal of MMP-9.

MMP 9 is produced by keratinocytes, monocytes, macrophages and PMN leukocytes. MMP9 is present in most cases of inflammatory

responses.

Principles of the Test

The kits is a solid sandwich enzyme-linked immunosorbent assay for detection of human MMP-9. Ananti-human MMP-9 monoclonal

antibody has been absorbed onto the wells of the microtiter strips provided. Samples including specimens or standards were pipetted into

wells. The human MMP-9 in specimens or standards would be captured by the coated antibody and the free others were removed by

washing. The human MMP-9 biotin-conjugated antibody were added and binds to human MMP-9 captured by the first antibody, which

formed a sandwich. Streptavidin-HRP would be added and binds to the biotin conjugated antibody, then free Streptavidin-HRP would be

removed during a wash step. After this, subtrate solution would be added and catalyzed by the HRP, and a coloured product is formed.

The intensity of the colored product is used to calculate in proportion to the amount of human MMP-9 in the original specimen.

Materials provided with the kits:

reagent 96/48Test Kit

Human MMP-9 Antibody-Coated Wells 12 strips/6 strips

Standard Diluent 10ml/5ml

Human MMP-9 Standard 2/1vial(s)

Human Detetion Antibody 10ml/5ml

Streptavidin-HRP 10ml/5ml

Wash Buffer Concentrate 20× 30ml/15ml

TMB 10ml/5 ml

Stop Solution 5ml/3 ml

Plate Covers 3/2

Complete Instruction Manual 1

Specimen Collection

1.Collecting specimen as following:

A.The particulate of the cell culture supernatants should be removed before use.

B.Serum was obtained from clot at room temperature.

C.Please collect plasma with EDTA.

D.Assay immediately or store samples at －20℃. Avoid free-thaw cycles.

2.Antiseptic and anticoagulant should not appear in Serum samples.

3.Any particulate should be removed from samples before use.4. Do not use grossly hemolyzed or lipemic samples.

Note: Strongly recommend that the serum and plasma samples should be diluent as doubling dilution before use.

Precautions for use:

1.Please storage the Kit at 2～8℃ 。

2. Washing buffer concentrate may have crystal in low temperature, and you can melt its in water-bath before use.

3. Please discard the remains after use of the dissolved standard.

4.Avoid contact of substrate solution with oxidizing agents and metal.

5.Usage of disposable pipette tips avoid microbial contamination or cross-contamination of reagents or specimens.

6. Do not mix or substitute reagents with those from other lots or other sources.

7. To ensure the adequate mixure of added reagents, please tap gently the plate after the wells were filled with liquid.

8. Incubation temperature should be 25～28℃.

9. Wash step was crucial for whole assay process.

10. Duplicate wells of the same sample were recommended in assay process.

11. Avoid the foam while pour the liquid into wells.

12.For serum or plasma samples ,the biotin-conjugated antibody should be incubate for at least 90 minutes.

Reagent Preparation

1.The reagents should be warmed up to room temperature before use. The remanent reagents must reseal and put into refrigeratory

again as soon as possible.

2.Dilute 1ml of wash buffer Concentrate into 19ml deionized or distilled water to work.

3. Dilute 1ml of 5×sample diluent into 4ml deionized or distilled water to 1×sample diluent.

4. Add the standard dilution solution to the bottle according to the volume of the label and wait15 minutes for complete dissolution.

Incubation temperature should be 25～28℃..And in turn add the half concentration diluent by standard diluent .

Wash step:

Automated microplate washer or operating by pipette: Each well should be pour into 300ul wash buffer and soak 15 or 30

seconds,then be aspirated, five times process were repeated. After the last wash, remove remaining wash buffer by aspirating.Invert the

plate and blot it against clean paper towels.

Materials Required But Not Provided

1. pipettes and pipette tips

2. Microwell strip reader capable of reading at 450 nm (540 nm as optional reference wave length)

3. automated microplate washer

4.Glass-distilled or deionized water

Assay procedure

1.The needed strips were putted into the frame, the remains were returned into foil pouch and resealed.

2.Blank well were recommended, which only color reagent and stop solution be added. It is suggested that each testing with gradient

density of standard for standard curve.

3.Add 100ul of standard or sample.Cover with the Plate Covers provided.Incubate for 2 hours at room temperature If is plasma serum

samples, different sample dilution ratio is different, generally in the range of 400 ~ 1500 times, if there is no definite scope, advice from

400 times dilution, if the sample concentration is too high, more than test scope, please increase after diluted times dilution test again.

4.Five times wash process were repeated.

5.Add 100ul of detetion antibody. Cover with the Plate Covers provided.Incubate for 1 hour at room temperature.

6.Five times wash process were repeated.

7.Add 100ul of Streptavidin-HRP. Cover with the Plate Covers provided. Lucifugal incubation for 20 minutes at room temperature.

8.Five times wash process were repeated.

9.Add 100ul of TMB ，Lucifugal incubation for 20 minutes at room temperature.

10.Add 50ul of stop solution to each well, determine the optical density of each well within 10 minutes.

Calculation of Results

上海尊龍凱時生物科技有限公司 www.ruianjituan.comHuman MMP-9 Standard Curve

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Human MMP-9 Concentration(pg/ml)

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上海尊龍凱時生物科技有限公司 www.ruianjituan.com

1.Duplicates should be within 20 per cent of the mean. Average absorbance values for each set of duplicate samples were used as

detection results.

2.The blank absorbance values of subtract should be deducted.

3.Drawing a best fit curve through the points of graph. Draw the standard curve by plotting assayed OD valure (on the Y axis) vs.

concentration (on the X axis). The sample concentration was obtained based on its OD value founding in the standard concentration

curve.

4.If the values obtained are not within the expected range of the standard, Samples should be dilute and assay again.

Typical Data and Standard Curve

concentration

(pg/ml)

Typical data 1 Typical data 2 Average

0 0.1273 0.1547 0.141

62.5 0.2852 0.295 0.2901

125 0.3438 0.3632 0.3535

250 0.5779 0.5966 0.58725

500 0.8491 0.8733 0.8612

1000 1.3001 1.3866 1.34335

2000 1.9914 2.304 2.1477

Human MMP-9 standard curve

Sensitivity, Specificity, Repeatability

Sensitivity: repeated assays were evalsuated and the minimum detectable dose was 7.3pg/ml.

Specificity : No significant cross-reactivity or interference with human MMP-1,MMP-2,MMP-3, MMP-7, MMP-8, MMP-10,

MMP-13,TIMP-2. TIMP-3. TIMP-4

Repeatability: The coefficient of variation between wells or plates is less than 10 percent.

REFERENCES:

1. Tamura, T. et al. (1996) Endocrinology 137:3729.

2. Okamoto, T. et al. (1997) J. Biol. Chem. 272:6059.

3. Cullen, B. et al. (1997) Int. J. Biochem. Cell Biol. 29:241.

4. Maeda, A. and R.A. Sobel (1996) J. Neuropathol. Exp. Neurol. 55:300.

5. Ueda, Y. et al. (1996) Am. J. Pathol. 148:611.

6. Ito, A. et al. (1996) J. Biol. Chem. 271:14657