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人的金屬蛋白酶 9 定量分析酶聯(lián)免疫檢測(cè)試劑盒

本試劑盒僅供科研使用。用于體外定量檢測(cè)人血清、血漿或細(xì)胞培養(yǎng)上清液中的 MMP-9 濃度。使用前請(qǐng)仔細(xì)閱讀說明書并檢查試劑組

分是否完整。如有產(chǎn)品包裝破損或質(zhì)量投拆，請(qǐng)?jiān)谑盏截浺粋€(gè)月之內(nèi)聯(lián)系我們。

MMP-9簡介：

MMPs蛋白是一組具有鋅指結(jié)構(gòu)的鈣依賴的能夠降解胞間基質(zhì)成分的一組蛋白的統(tǒng)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(jié)構(gòu)上，MMP-9的催化區(qū)包括3個(gè)重復(fù)的型纖維連接蛋白結(jié)構(gòu)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(jié)構(gòu)域和含鋅指結(jié)構(gòu)的結(jié)構(gòu)域，MMP-9包含一個(gè)V型的膠原蛋白結(jié)構(gòu)域,這個(gè)結(jié)構(gòu)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(nèi)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

組織抑制因子1（TIMP-1）作為MMP-9的抑制劑，與MMP-9的酶原或活化后酶的催化區(qū)的羧末端特異性結(jié)合，形成復(fù)合物。

MMP-9可由角化細(xì)胞、單核細(xì)胞、巨噬細(xì)胞和多形核白細(xì)胞等產(chǎn)生，在一些炎性反應(yīng)的過程中常伴隨著MMP-9的產(chǎn)生。

檢測(cè)原理:

本試劑盒采用雙抗體夾心ELISA法檢測(cè)樣本中MMP-9 的濃度。MMP9 捕獲抗體已預(yù)包被于酶標(biāo)板上，當(dāng)加入標(biāo)本或參考品時(shí)，其中的

MMP-9 會(huì)與捕獲抗體結(jié)合，其它游離的成分通過洗滌的過程被除去。當(dāng)加入生物素化的抗人MMP-9 抗體后，抗人MMP-9 抗體與MMP-9 接合，

形成夾心的免疫復(fù)合物，其它游離的成分通過洗滌的過程被除去。隨后加入辣根過氧化物酶標(biāo)記的親合素。生物素與親合素特異性結(jié)合，

親合素連接的酶就會(huì)與夾心的免疫復(fù)合物連接起來；其它游離的成分通過洗滌的過程被除去。最后加入顯色劑，若樣本中存在MMP-9 將會(huì)

形成免疫復(fù)合物，辣根過氧化物酶會(huì)催化無色的顯色劑氧化成藍(lán)色物質(zhì)，在加入終止液后呈黃色。通過酶標(biāo)儀檢測(cè)，讀其450nm處的OD值，

MMP-9 濃度與OD450值之間呈正比，通過參考品繪制標(biāo)準(zhǔn)曲線，對(duì)照未知樣本中OD值，即可算出標(biāo)本中MMP-9 濃度。

人MMP-9定量分析酶聯(lián)免疫檢測(cè)試劑盒組成:

組分 規(guī)格（96T/48T）

人MMP-9預(yù)包被板 12條/6條

5X標(biāo)準(zhǔn)品稀釋液 10ml/5ml

人MMP-9標(biāo)準(zhǔn)品 2/1支(凍干)

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(biāo)本收集:

1.標(biāo)本的收集請(qǐng)按下列流程進(jìn)行操作；

A.細(xì)胞上清標(biāo)本離心去除懸浮物后即可；

B.血清標(biāo)本應(yīng)是自然凝固后，取上清，避免在冰箱中凝固血液；

C.血漿標(biāo)本，推薦用EDTA的方法收集；

D.標(biāo)本收集后請(qǐng)分裝，若待測(cè)樣本不能及時(shí)檢測(cè)，凍存于－20℃，避免反復(fù)凍融。

2.血清標(biāo)本不應(yīng)添加任何防腐劑或抗凝劑；

3.標(biāo)本應(yīng)清澈透明，檢測(cè)前樣本中如有懸浮物應(yīng)通過離心去除。

4.請(qǐng)勿使用溶血高血脂或污染的標(biāo)本檢測(cè)，否則結(jié)果將不準(zhǔn)確。注：人血清或血漿樣本請(qǐng)用標(biāo)準(zhǔn)品稀釋液稀釋后再檢測(cè)。

注意事項(xiàng):

1.試劑盒請(qǐng)保存在2～8℃。

2.濃縮洗滌液因在低溫下可能有結(jié)晶，請(qǐng)水浴加熱使結(jié)晶完全溶解后再配制工作液。

3.標(biāo)準(zhǔn)品復(fù)溶加樣后，剩余部份請(qǐng)丟棄。

4.底物請(qǐng)勿接觸氧化劑和金屬。

5.加樣時(shí)，請(qǐng)及時(shí)更換槍頭，避免交叉污染。

6.嚴(yán)禁混用不同批號(hào)的試劑盒組份。

7.充分混勻?qū)ΡＷC反應(yīng)結(jié)果的準(zhǔn)性很重要，在加液后請(qǐng)輕輕叩擊邊緣以保證混勻。

8.室溫反應(yīng)，請(qǐng)嚴(yán)格控制在25～28℃。

9.洗滌過程是至關(guān)重要的，洗滌不充分會(huì)使精確度下降并導(dǎo)致結(jié)果誤差較大。

10.試驗(yàn)中標(biāo)準(zhǔn)品和樣本檢測(cè)時(shí)建議作雙復(fù)孔。

11.加樣過程中避免氣泡的產(chǎn)生。

12.血清和血漿標(biāo)本的檢測(cè)時(shí)，檢測(cè)抗體的孵育時(shí)間應(yīng)適當(dāng)延長。

檢測(cè)前準(zhǔn)備工作:

1.試劑盒自冰箱中取出后應(yīng)置室溫（25～28℃）平衡20分鐘；每次檢測(cè)后剩余試劑請(qǐng)及時(shí)于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(biāo)準(zhǔn)品稀釋液用雙蒸水或去離子水稀釋(1份加4份水)。

4.標(biāo)準(zhǔn)品: 按標(biāo)簽復(fù)溶體積加入1X標(biāo)準(zhǔn)品稀釋液復(fù)溶使MMP-9終濃度達(dá)到2000pg/ml，室溫反應(yīng)，請(qǐng)嚴(yán)格控制在25～28℃，靜置15~20分鐘后

輕輕混懸（建議抽吸幾次）待徹底溶解，用標(biāo)準(zhǔn)品稀釋液倍比梯度稀釋后依次加入檢測(cè)孔中。（標(biāo)準(zhǔn)曲線取七個(gè)點(diǎn)，最高濃度為2000 pg/ml,

標(biāo)準(zhǔn)品稀釋液直接加入作為0濃度.）

洗滌方法:

自動(dòng)洗板機(jī)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。最后一次洗板完成后將板倒扣著在厚吸水紙上用力拍干。

實(shí)驗(yàn)過程需自備的材料：

1.不同規(guī)格的加樣槍及相應(yīng)的槍頭；

2.酶標(biāo)儀；

3.自動(dòng)洗板機(jī)；

4.去離子水或雙蒸水；

操作步驟:

1.通過計(jì)算并確定一次性實(shí)驗(yàn)所需的板條數(shù)，取出所需板條放置在框架內(nèi)，暫時(shí)用不到板條請(qǐng)放回鋁箔袋密封，保存于4℃。

2.建議設(shè)置本底較正孔，即空白孔,設(shè)置方法為該孔只加TMB顯色液和中止液。每次實(shí)驗(yàn)均需做標(biāo)準(zhǔn)品對(duì)照并畫出標(biāo)準(zhǔn)曲線。

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

本，不同樣本稀釋比例不一樣，一般范圍在 400~1500 倍，如無明確范圍，建議從 400 倍稀釋，如果樣本濃度過高，超過檢測(cè)范圍，請(qǐng)加大

稀釋倍數(shù)后重新稀釋檢測(cè)。

4.洗板5次，且最后一次置厚吸水紙上拍干。

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

6.洗板5次，且最后一次置厚吸水紙上拍干。

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(yīng)孔，避光室溫（25～28℃）孵育20分鐘。

上海傳秋生物科技有限公司 424100.com.cn人MMP-9標(biāo)準(zhǔn)曲線

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人MMP-9濃度（pg/ml）

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上海傳秋生物科技有限公司 424100.com.cn

8.洗板5次，且最后一次置厚吸水紙上拍干。

9.加入顯色劑TMB100ul/孔，避光室溫（25～28℃）孵育20分鐘。

10.加入終止液50ul/孔,混勻后即刻測(cè)量OD450值。

結(jié)果判斷:

1.復(fù)孔的值在20%的差異范圍內(nèi)結(jié)果才有效，復(fù)孔的值平均后可作為測(cè)量值。

2.每個(gè)標(biāo)準(zhǔn)品或標(biāo)本的OD值應(yīng)減去本底校正孔的OD值。

3.手工繪制標(biāo)準(zhǔn)曲線。以標(biāo)準(zhǔn)品濃度作橫坐標(biāo)，OD值作縱坐標(biāo)，以平滑線連接各標(biāo)準(zhǔn)品的坐標(biāo)點(diǎn)。通過標(biāo)本的OD值可在標(biāo)準(zhǔn)曲線上查出其

濃度。

4.若標(biāo)本 OD 值高于標(biāo)準(zhǔn)曲線上限，應(yīng)適當(dāng)稀釋后重測(cè)，計(jì)算濃度時(shí)應(yīng)乘以稀釋倍數(shù)。

典型數(shù)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(biāo)準(zhǔn)曲線

注意：本圖僅供參考，應(yīng)以同次試驗(yàn)標(biāo)準(zhǔn)品所繪標(biāo)準(zhǔn)曲線計(jì)算標(biāo)本含量。

靈敏度，特異性和重復(fù)性:

1.靈敏度：多次重復(fù)結(jié)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(yīng)。

3.重復(fù)性：板內(nèi)，板間變異系數(shù)均<10%.

參考文獻(xiàn):

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.上海傳秋生物科技有限公司 424100.com.cn

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

上海傳秋生物科技有限公司 424100.com.cnHuman MMP-9 Standard Curve

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Human MMP-9 Concentration(pg/ml)

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上海傳秋生物科技有限公司 424100.com.cn

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 evaluated 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.

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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