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大鼠血管內皮生長因子定量分析酶聯免疫檢測試劑盒

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

分是否完整, 本試劑盒僅供科研使用 。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 。

VEGF 簡介 ：

VEGF屬於PDGF因子家族 ，是A 、B 、C 、D四種亞類的一類因子的總稱 ，它們在血管發生過程中起重要作用 。其中在血管再生和內皮細胞

的再生過程中 ，VEGF-A扮演重要的角色 。現已發現VEGF-A有許多種因切割後氨基酸數不同的亞型 ，其中含有的肝磷脂接合區域 ，能與胞外

基質內的肝磷脂結合 ，以VEGF165量最多 。VEGF-A可由多種細胞分泌的分子量為45kDa的糖蛋白 。

VEGF的功能主要表現在傷口的愈合和雌性生殖周期循環上 。在病理組織中 ，VEGF能提升血管通透性 。這就是腫瘤的轉移和腫瘤複發的

基礎 。在胚胎期 ，VEGF的作用表現為調控胚胎細胞的增殖 、轉移和提高內皮細胞的存活力上 ，通過成骨細胞和成軟骨細胞的驀集作用提升

骨形成的效率 。VEGF-A的功能還表現在一些如血管再生和血管通透相關聯的生理過程中 。腫瘤釋放的細胞因子和生長因子能刺激骨髓中的

巨核細胞分裂為血小板 ，這樣就間接增加了VEGF-A向腫瘤的遷移 。骨組織 、心肌 、肝實質 、成骨細胞 、中性粒細胞 、巨噬細胞 、角化細胞 、

棕色脂肪組織 、CD34+幹細胞 、內皮細胞 、成纖維細胞和血管平滑肌細胞等細胞和組織均可表達 VEGF 。

檢測原理:

本試劑盒采用雙抗體夾心ELISA法檢測樣本中大鼠VEGF的濃度 。大鼠VEGF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

的大鼠VEGF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大鼠VEGF抗體後 ，抗大鼠VEGF抗體與大鼠VEGF

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VEGF

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 ，大鼠VEGF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VEGF濃度 。

大鼠VEGF定量分析酶聯免疫檢測試劑盒組成:

組分 規格（96T/48T）

大鼠VEGF預包被板 12條/6條

樣本分析緩衝液 5ml/3ml

標準品稀釋液 10ml/5ml

大鼠VEGF標準品 4支/2支(凍幹)*

大鼠VEGF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.若待測樣本不能及時檢測 ，標本收集後請分裝 ，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 ：大鼠血清或血漿樣本請用樣本分析緩衝液做倍比稀釋後再檢測 。

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.如有5X準品稀釋液 ，請按所需量用雙蒸水或去離子水稀釋(1份加4水) 。

4.標準品: 按標簽複溶體積加入標準品稀釋液複溶使VEGF終濃度達到1000pg/ml ，室溫反應 ，請嚴格控製在25～28℃ ，靜置10~15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1000pg/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 。對於血清或血漿標本 ，

請加入50 ul樣本分析緩衝液後加50 ul標本 ，如稀釋量大 ，請將樣本與樣本分析緩衝液等量加入 ，不足部分用標準品稀釋液補充至100ul 。

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.加入親和素鏈接的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大鼠VEGF標準曲線

0

0.5

1

1.5

2

2.5

0 31.25 62.5 125 250 500 1000

大鼠VEGF 濃度（pg/ml）

O

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 0.1084 0.1264 0.1174

31.25 0.0217 0.3663 0.194

62.5 0.1987 0.4507 0.3247

125 0.8126 0.49002 0.65131

250 1.1253 0.8931 1.0092

500 1.2861 1.5371 1.4116

1000 2.1955 1.9975 2.0965

大鼠VEGF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14pg/ml 。

2.特異性：與GDNF, GM-CSF,TNF-α ,IL-1α ,IL-1β ,IL-2 ,IL-4 ,PDGF-BB, IL-10,人 VEGF,小鼠VEGF

等沒有交叉反應 。

3.重複性 ：板內 ，板間變異係數均<10%.

參考文獻 ：

1. Clauss, M. (2000) Semin. Thromb. Hemost. 26:561.

2. Shibuya, M. (2001) Cell Struct. Funct. 26:25.

3. Woolard, J. et al. (2004) Cancer Res. 64:7822.

4. Yamane, A. et al. (1994) Oncogene 9:2683.

5. Namiki, A. et al. (1995) J. Biol. Chem. 270:31189.

6. Fuh, G. et al. (2000) J. Biol. Chem. 275:26690.上海尊龍凱時生物科技有限公司 www.ruianjituan.com

ELISA Kit for the Quantitative Analysis of Rat VEGF

The rat VEGF ELISA (enzyme-linked immunosorbent assay) kit is used for detection of rat VEGF in cell culture supernatants, rat

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

VEGF with several members including VEGF-A, VEGF-B,VEGF-C- and VEGF-D, belonging to PDGF family, is best known for its

role in vasculogenesis. VEGF-A is one of the key molecules for angiogenesis and the survival of the endothelium. Many different splice

variants of VEGF-A have been described, in which VEGF165 is the most predominant protein and anchors with its heparin binding

domain to extracellular matrix haparin. VEGF-A is a glycoprotein, secreted as a homodimer of 45 kDa by many different cell types. VEGF

functions mainly in wound healing and the female reproductive cycle. In diseased tissues, VEGF promotes vascular permeability. It is

thus thought to contribute to tumor metastasis by promoting both extravasation and tumor angiogenesis. During embryogenesis, VEGF

regulates the proliferation, migration, and survival of endothelial cells and promotes bone formation through osteoblast and chondroblast

recruitment. VEGF-A is implicated in several other pathological conditions associated with enhancing angiogenesis or vascular

permeability. Tumors can release cytokines and growth factors that stimulate the production of megakaryocytes in the marrow and

elevate the platelet count. This can result in indirect increase of VEGF-A delivery to tumors.VEGF is expressed in multiple cells and

tissues including skeletal and cardiac muscle, hepatocytes, osteoblasts , neutrophils , macrophages , keratinocytes , brown adipose

tissue , CD34+ stem cells , endothelial cells , fibroblasts, and vascular smooth muscle cells .

Principles of the Test

The kits is a solid sandwich enzyme-linked immunosorbent assay for detection of rat VEGF. An anti-rat VEGF monoclonal

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

wells. The rat VEGF in specimens or standards would be captured by the coated antibody and the free others were removed by washing.

The rat VEGF biotin-conjugated antibody were added and binds to rat VEGF 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 mouse VEGF in the original specimen.

Materials provided with the kits:

reagent 96/48Test Kit

Assay Buffer 5ml/3ml

Rat VEGF Antibody-Coated Wells 12 strips/6 strips

Standard Diluent 10ml/5ml

Rat VEGF Standard 4/2vial(s)

Rat VEGF 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℃. 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. If you have a 5x standard diluent, please dilute it with double steaming water or deionized water.

4. Add standard diluent to the bottle according to the volume of the label and wait 15 minutes for complete dissolution. 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 into300ul 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 assay the serum

sample,you should add 50μ l assay buffer with 50μ l sample into the wells,if the protein concentration is higher than the range of the Kit,

add the same quantitys assay buffer with the sample, the deficiency should be complemented with sample diluent to 100μ l per well.

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 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 0.1084 0.1264 0.1174

31.25 0.0217 0.3663 0.194

62.5 0.1987 0.4507 0.3247

125 0.8126 0.49002 0.65131

250 1.1253 0.8931 1.0092

500 1.2861 1.5371 1.4116

1000 2.1955 1.9975 2.0965

Rat VEGF standard curve

Sensitivity,Specificity, Repeatability

Sensitivity: repeated assays were evalsuated and the minimum detectable dose was 14pg/ml.

Specificity: No significant cross-reactivity or interference with GDNF, GM-CSF,TNF-α ,IL-1α ,IL-1β , IL-2 , IL-4 , PDGF-BB,

IL-10,human VEGF, mouse VEGF.

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

REFERENCES:

1. Clauss, M. (2000) Semin. Thromb. Hemost. 26:561.

2. Shibuya, M. (2001) Cell Struct. Funct. 26:25.

3. Woolard, J. et al. (2004) Cancer Res. 64:7822.

4. Yamane, A. et al. (1994) Oncogene 9:2683.

5. Namiki, A. et al. (1995) J. Biol. Chem. 270:31189.

6. Fuh, G. et al. (2000) J. Biol. Chem. 275:26690