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人血管生成素定量分析酶聯(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)上清液中的血管生成素濃度。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。

人血管生成素簡(jiǎn)介：

人血管生成素是一種非糖基化的多肽，有 123 個(gè)氨基酸殘基組成，分子量為 14 kDa，含有三個(gè)鏈內(nèi)二硫鍵，屬于核糖核酸酶家族。

血管生成素一般由血管內(nèi)皮細(xì)胞、平滑肌細(xì)胞、成纖維細(xì)胞、結(jié)腸柱狀上皮細(xì)胞、淋巴細(xì)胞、腺癌干細(xì)胞以及腫瘤細(xì)胞系等細(xì)胞產(chǎn)生。

血管生成素通常以分泌蛋白形式進(jìn)入血液中，其含量高達(dá) 60-120 ng / mL。

血管生成素作為核糖核酸酶與其它的核糖核酸酶家族成員一樣，具有細(xì)胞毒性，其機(jī)理主要是抑制細(xì)胞中蛋白質(zhì)的合成從而對(duì)細(xì)胞具

有毒性。血管生成素也可以通過磷脂酶的活化，刺激毛細(xì)血管和臍靜脈內(nèi)皮細(xì)胞生成甘油二酯以及前列腺素的分泌。

檢測(cè)原理:

本試劑盒采用雙抗體夾心 ELISA 法檢測(cè)樣本中血管生成素的濃度。血管生成素捕獲抗體已預(yù)包被于酶標(biāo)板上，當(dāng)加入標(biāo)本或參考品時(shí)，

其中的血管生成素會(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血管生成素抗體后，抗人血管生成

素抗體與血管生成素接合，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，

若樣本中存在血管生成素將會(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，血管生成素濃度與 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

中血管生成素濃度。

人血管生成素定量分析酶聯(lián)免疫檢測(cè)試劑盒組成:

組分 規(guī)格（96T/48T）

人血管生成素預(yù)包被板 12條/6條

5×標(biāo)準(zhǔn)品稀釋液 20ml/10ml

人血管生成素標(biāo)準(zhǔn)品 8支/4支(凍干)

人血管生成素生物素化抗體 10ml/5ml

親和素連接的HRP酶 10ml/5ml

濃縮洗滌液 20× 30ml/15ml

TMB底物 10ml/5ml

中止液 5ml/3ml

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.若待測(cè)樣本不能及時(shí)檢測(cè)，標(biāo)本收集后請(qǐng)分裝，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(zhǎng)

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)溶使血管生成素終濃度達(dá)到500pg/ml，室溫反應(yīng)，請(qǐng)嚴(yán)格控制在25～28℃，靜置10~15

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300

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。對(duì)于血清或血漿標(biāo)本，

請(qǐng)用1×標(biāo)準(zhǔn)品稀釋液稀釋至合適濃度檢測(cè)，建議稀釋100~8000倍。

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。

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.加入中止液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。

上海傳秋生物科技有限公司 424100.com.cn血管生成素參考標(biāo)準(zhǔn)曲線

0

0.5

1

1.5

2

2.5

0 15.625 31.25 62.5 125 250 500

濃度（pg/ml）

O

D值

上海傳秋生物科技有限公司 424100.com.cn

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 0.064 0.082 0.073

15.625 0.217 0.253 0.235

31.25 0.427 0.451 0.439

62.5 0.703 0.803 0.753

125 1.025 1.123 1.074

250 1.486 1.61 1.548

500 2.066 2.282 2.174

人Angiogenin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4.75pg/ml。

2.特異性：與EGF，GM-CSF，MIP-1α ，MIP-1β ，TGF-α ，TGF-β 1,小鼠 EGF，GM-CSF，MIP-1α 等沒有交叉反應(yīng)。

3.重復(fù)性：板內(nèi)，板間變異系數(shù)均<10%.

參考文獻(xiàn):

1. Moroianu, J. and J.F. Riordan (1994) Proc. Natl. Acad. Sci. USA 91:1677.

2. Moenner, M. et al. (1994) Eur. J. Biochem. 226:483.

3. Bond, M.D. et al. (1993) Biochem. Biophys. Acta 1162:177.

4. Beintema, J.J. (1989) FEBS Lett. 254:1.

5. Hu, G-F. et al. (1991) Proc. Natl. Acad. Sci. USA 88:2227.

6. Russo, N. et al. (1994) Proc. Natl. Acad. Sci. USA 91:2920.上海傳秋生物科技有限公司 424100.com.cn

ELISA Kit for the Quantitative Analysis of Human Angiogenin

The human IL-6 ELISA (enzyme-linked immunosorbent assay) kit is used for detection of human IL-6 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

Human Angiogenin (ANG) is 123 amino acid (aa) residues in length and contains three intra-chain disulfide bonds, 14

kDa, non-glycosylated polypeptide that is classified as a member of the ribonuclease superfamily.

Cells known to express ANG include vascular endothelial cells, smooth muscle cells, fibroblasts, colonic columnar

epithelium, lymphocytes, and primary adenocarcinoma cells as well as select tumor cell lines. Angiogenin is present in

normal human plasma at levels as high as 60-120 ng/mL.

Similar to several members of the ribonuclease superfamily, Angiogenin is a cytotoxic agent that can abolish cellular

protein synthesis. However, Angiogenin can stimulate capillary and umbilical vein endothelial cells to produce

diacylglycerol and secrete prostacyclin by phospholipase activation.

Principles of the Test

The kits is a solid sandwich enzyme-linked immunosorbent assay for detection of human IL-6. An anti-human IL-6

monoclonal antibody has been absorbed onto the wells of the microtiter strips provided.

Samples including specimens or standards were pipetted into wells. The human ANGIOGENIN in specimens or standards would be

captured by the coated antibody and the free others were removed by washing. The human ANGIOGENIN biotin-conjugated antibody

were added and binds to human ANGIOGENIN 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 ANGIOGENIN in the original specimen.

Materials provided with the kits:

Reagent 96/48Test Kit

Human Angiogenin Antibody-Coated Wells 12 strips/6 strips

5×Standard Diluent 20 ml/10ml

Human Angiogenin Standard 8/4vial(s)

Human Angiogenin Detetion Antibody 10ml/5ml

Streptavidin-HRP 10ml/5ml

Wash Buffer Concentrate 20× 30ml/15ml

TMB 10ml/5ml

Stop Solution 5ml/3ml

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℃.Avoidfree-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 deionized or distilled water 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 temperatureIf assay the serum

sample, you should diluent the sample to proper times by experiment ,we sagest 100~8000 folds diluent before detection

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

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

detection results.

上海傳秋生物科技有限公司 424100.com.cnAngiogenin Standard Curve

0

0.5

1

1.5

2

2.5

0 15.625 31.25 62.5 125 250 500

human Angiogenin Concentration（pg/ml）

O

p

t

i

c

a

l

D

e

n

s

i

t

y

上海傳秋生物科技有限公司 424100.com.cn

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

concentratio

n (pg/ml)

Typical data 1 Typical data 2 Average

0 0.064 0.082 0.073

15.625 0.217 0.253 0.235

31.25 0.427 0.451 0.439

62.5 0.703 0.803 0.753

125 1.025 1.123 1.074

250 1.486 1.61 1.548

500 2.066 2.282 2.174

Human Angiogenin Standard Curve

Sensitivity, Specificity, Repeatability

Sensitivity: repeated assays were evaluated and the minimum detectable dose was 4.75pg/ml.

Specificity: No significant cross-reactivity or interference with humanEGF，GM-CSF，MIP-1α ，MIP-1β ，TGF-α ，TGF-β 1,mouse EGF，

GM-CSF，MIP-1α

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

REFERENCES:

1. Moroianu, J. and J.F. Riordan (1994) Proc. Natl. Acad. Sci. USA 91:1677.

2. Moenner, M. et al. (1994) Eur. J. Biochem. 226:483.

3. Bond, M.D. et al. (1993) Biochem. Biophys. Acta 1162:177.

4. Beintema, J.J. (1989) FEBS Lett. 254:1.

5. Hu, G-F. et al. (1991) Proc. Natl. Acad. Sci. USA 88:2227.

6. Russo, N. et al. (1994) Proc. Natl. Acad. Sci. USA 91:2920.