人VEGF ELISA试剂盒

人VEGF定量分析酶联免疫检测试剂盒

本试剂盒仅供科研使用。用于体外定量检测人血清、血浆或细胞培养上清液中的VEGF浓度。使用前请仔细阅读说明书并检查试剂组分是否完整,如有疑问请与北京博凌科为生物科技有限公司联系，我们将提供力所能及的帮助。如您有其它需求，请登录北京博凌科为生物科技有限公司网站或致电本公司。

VEGF简介：

VEGF属于PDGF因子家族，是A、B、C、D四种亚类的一类因子的总称，它们在血管发生过程中起重要作用。其中在血管再生和内皮细胞的再生过程中，VEGF-A扮演重要的角色。现已发现VEGF-A有许多种因切割后氨基酸数不同的亚型，其中含有的肝磷脂接合区域，能与胞外基质内的肝磷脂结合，以VEGF165量最多。VEGF-A可由多种细胞分泌的分子量为45kDa的糖蛋白。

VEGF的功能主要表现在伤口的愈合和女性生殖周期循环上。在病理组织中，VEGF能提升血管通透性。这就是肿瘤的转移和肿瘤复发的基础。在胚胎期，VEGF的作用表现为调控胚胎细胞的增殖、转移和提高内皮细胞的存活力上，通过成骨细胞和成软骨细胞的蓦集作用提升骨形成的效率。VEGF-A的功能还表现在一些如血管再生和血管通透相关联的生理过程中。肿瘤释放的细胞因子和生长因子能刺激骨髓中的巨核细胞分裂为血小板，这样就间接增加了VEGF-A向肿瘤的迁移。

骨组织、心肌、肝实质、成骨细胞、中性粒细胞、巨噬细胞、角化细胞、棕色脂肪组织、CD34+干细胞、内皮细胞、成纤维细胞和血管平滑肌细胞等细胞和组织均可表达VEGF。

检测原理:

本试剂盒采用双抗体夹心ELISA法检测样本中VEGF的浓度。VEGF捕获抗体已预包被于酶标板上，当加入标本或参考品时，其中的VEGF会与捕获抗体结合，其它游离的成分通过洗涤的过程被除去。当加入生物素化的抗人VEGF抗体后，抗人VEGF抗体与VEGF接合，形成夹心的免疫复合物，其它游离的成分通过洗涤的过程被除去。随后加入辣根过氧化物酶标记的亲合素。生物素与亲合素特异性结合，亲合素连接的酶就会与夹心的免疫复合物连接起来；其它游离的成分通过洗涤的过程被除去。最后加入显色剂，若样本中存在VEGF将会形成免疫复合物，辣根过氧化物酶会催化无色的显色剂氧化成蓝色物质，在加入终止液后呈黄色。通过酶标仪检测，读其450nm处的OD值，VEGF 浓度与OD450值之间呈正比，通过参考品绘制标准曲线，对照未知样本中OD值，即可算出标本中VEGF 浓度。

人定量分析酶联免疫检测试剂盒组成:

组分规格

预包被板12条或6条

样本分析缓冲液1瓶5ml/3ml

标准品稀释液10ml/5ml

标准品2/1支(冻干)*

生物素化抗体1瓶10ml/5ml

HRP连接的酶结合物1瓶10ml/5ml

浓缩洗涤液20×30ml/瓶

TMB底物1瓶10ml/5ml

终止液1瓶5ml/3ml

封板胶纸3/2张

说明书1份

标本收集:

1.标本的收集请按下列流程进行操作；

A.细胞上清标本离心去除悬浮物后即可；

B.血清标本应是自然凝固后，取上清，避免在冰箱中凝固血液；

C.血浆标本，推荐用EDTA的方法收集；

D.若待测样本不能及时检测，标本收集后请分装，冻存于－20℃，避免反复冻融。

2.血清标本不应添加任何防腐剂或抗凝剂；

3.标本应清澈透明，检测前样本中如有悬浮物应通过离心去除。

4.请勿使用溶血，高血脂或污染的标本检测，否则结果将不准确。

注：正常人血清或血浆样本请用标本缓冲液做倍比稀释后再检测。

注意事项:

1.试剂盒请保存在2～8℃。

2.浓缩洗涤液因在低温下可能有结晶，请水浴加热使结晶完全溶解后再配制工作液。

3.若标准品复溶后，请在三天内用完。

4、底物请勿接触氧化剂和金属。

5、加样时，请及时更换枪头，避免交叉污染。

6.严禁混用不同批号的试剂盒组份。

7.充分混匀对保证反应结果的准性很重要，在加液后请轻轻叩击边缘以保证混匀。

8.室温反应，请严格控制在25～28℃。

9.洗涤过程是至关重要的，洗涤不充分会使精确度下降并导致结果误差较大。

10.试验中标准品和样本检测时建议作双复孔。

11.加样过程中避免气泡的产生。

12、血清和血浆标本的检测时，检测抗体的孵育时间应适当延长。

检测前准备工作:

1.试剂盒自冰箱中取出后应置室温平衡20分钟；每次检测后剩

余试剂请及时于2～8℃保存。

2.将浓缩洗涤液用双蒸水或去离子水稀释(1份加19份水)。

3.标准品:加入去离子水0.5ml至冻干标准品瓶中，静置几分钟后轻轻混悬待彻底溶解，用标准品稀释液倍比梯度稀释后依次加入检测孔中。（0浓度即标准品稀释液直接加入）

洗涤方法:

自动洗板机或人工洗板：每孔洗涤液为400ul，注入与吸出间隔15-30秒。洗板5次。最后一次洗板完成后将板倒扣着在厚吸水纸上用力拍干。

实验过程需自备的材料：

1.不同规格的加样枪及相应的枪头；

2.酶标仪；

3．自动洗板机；4．去离子水或双蒸水；

操作步骤:

1.通过计算并确定一次性实验所需的板条数，取出所需板条放置在框架内，暂时用不到板条请放回铝箔袋密封，保存于4℃。

2.建议设置本底较正孔，即空白孔,设置方法为该孔只加TMB显色液和中止液。每次实验均需做标准品对照并画出标准曲线。

3.分别将标本或不同浓度标准品(100ul/孔)加入相应孔中，用封板胶纸封住反应孔，室温孵育120分钟。对于血清或血浆标本，请加入50ul样本分析缓冲液后加50ul标本。

4.洗板5次，且最后一次置厚吸水纸上拍干。

5.加入生物素化抗体工作液(100ul/孔)。用封板胶纸封住反应孔，室温孵育60分钟。

6.洗板5次，且最后一次置厚吸水纸上拍干。

7.加入酶结合物工作液(100ul/孔)。用封板胶纸封住反应孔，避光室温孵育20分钟。

8.洗板5次，且最后一次置厚吸水纸上拍干。

9.加入显色剂TMB100ul/孔，避光室温孵育20分钟。

10.加入终止液50ul/孔,混匀后即刻测量OD450值。

结果判断:

1.复孔的值在20%的差异范围内结果才有效，复孔的值平均后可作为测量值。

2.每个标准品或标本的OD值应减去本底校正孔的OD值。

3.手工绘制标准曲线。以标准品浓度作横坐标，OD值作纵坐标，以平滑线连接各标准品的坐标点。通过标本的OD值可在标准曲线上查出其浓度。

4.若标本OD值高于标准曲线上限，应适当稀释后重测，计算浓度时应乘以稀释倍数。

典型数值和参考曲线

浓度pg/ml 典型OD值

1

典型OD值

2

OD平均值

00.11070.12010.1154

62.50.24770.31660.28215

1250.34170.39260.36715

2500.58860.62690.60775

5000.82840.87840.8534

1000 1.3247 1.4743 1.3995

2000 1.9437 2.0386 1.99115

人VEGF参考标准曲线

灵敏度，特异性和重复性:

1.灵敏度：多次重复结果表明，最小检出量为3.7pg/ml。

2.特异性：与人PLGF、VEGF-C、VEGF-D、小鼠PLGF-2、VEGF120、VEGF164和大鼠VEGF164等没有交叉反应。

3.重复性：板内，板间变异系数均<10%.

参考文献:

Robinson,C.J.and S.E.Stringer(2001)J.Cell.Sci.114:853.

Richardson,R.S.et al.(1999)Am.J.Physiol.277:H2247.

Mclaren,J.et al.(1996)J.Clin.Invest.98:482.

Bautz,F.et al.(2000)Exp.Hematol.28:700.

ELISA Kit for the Quantitative Analysis of Human VEGF

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

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 VEGF165is the most predominant protein and anchors with its heparin binding domain to extracellular matrix haparin.VEGF-A is a glycoprotein,secreted as a homodimer of45kDa 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 human VEGF.An anti-human VEGF monoclonal antibody has been absorbed onto the wells of the microtiter strips provided.Samples including specimens or standards were pipetted into wells.The human VEGF in specimens or standards would be captured by the coated antibody and the free others were removed by washing.The human VEGF biotin-conjugated antibody were added and binds to human 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 human VEGF in the original specimen.

Materials provided with the kits:

reagent96/48Test Kit

Assay Buffer5ml/3ml

VEGF Antibody-Coated Wells12strips/6strips

Standard Diluent10ml/5ml

VEGF Standard2/1vial(s)

Detetion Antibody10ml/5ml

Streptavidin-HRP10ml/5ml

Wash Buffer Concentrate20×30ml

TMB10ml/5ml

Stop Solution5ml/3ml

Plate Covers3/2

Complete Instruction Manual1

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

Precautions for use:

1.Please storage the Kit at2～8℃。

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

3.Please discard the dissolved standard after3days for use.

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 be25～28℃.

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 least90minutes.

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.Dilute1ml of wash buffer Concentrate into19ml deionized or distilled water to work.

3Add0.5ml deionized or distilled water to bottle wait 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 into400ul wash buffer and soak15or30seconds,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 at450nm(540nm 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.Add100ul of standard or sample.Cover with the Plate Covers provided.Incubate for2hours at room temperature.

4.Five times wash process were repeated.

5.Add100ul of detetion antibody.Cover with the Plate Covers provided.Incubate for1hour at room temperature.

6.Five times wash process were repeated.

7.Add100ul of Streptavidin-HRP.Cover with the Plate Covers provided.Lucifugal incubation for20minutes at room temperature.

8.Five times wash process were repeated.

9.Add100ul of TMB，Lucifugal incubation for20minutes at room temperature.

10.Add50ul of stop solution to each well,determine the optical density of each well within10minutes.

Calculation of Results

1.Duplicates should be within 20per 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.

T ypical Data and S tandard C urve

concentratio

n(pg/ml)

T ypical data 1T ypical data 2A verage 0

0.11070.12010.115462.5

0.24770.31660.28215125

0.34170.39260.36715250

0.58860.62690.60775500

0.82840.87840.85341000

1.3247 1.4743 1.39952000 1.9437

2.0386

1.99115

H uman VEGF standard curve

specificity,Repeatability

Sensitivity:repeated assays were evaluated and the minimum detectable dose was 3.7pg/ml.specificity :No significant cross-reactivity or interference with humanPLGF ，VEGF-C ，VEGF-D ，Mouse PLGF-2，VEGF120，VEGF164and rat VEGF164.

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

Robinson,C.J.and S.E.Stringer (2001)J.Cell.Sci.114:853.

Richardson,R.S.et al.(1999)Am.J.Physiol.277:H2247.

Mclaren,J.et al.(1996)J.Clin.Invest.98:482. Bautz,F.et al.(2000)Exp.Hematol.28:700.