猪血管内皮细胞生长因子（VEGF）酶联免疫分析(ELISA)
试剂盒使用说明书
本试剂仅供研究使用       目的：本试剂盒用于测定猪血清、血浆、组织
匀浆及相关液体样本中血管内皮细胞生长因子（VEGF）的含量。
 
实验原理：
本试剂盒应用双抗体夹心法测定标本中猪血管内皮细胞生长因子（VEGF）水平。用纯
化的猪血管内皮细胞生长因子（VEGF）捕获抗体包被微孔板，制成固相抗体，往包被的微孔
中依次加入猪血管内皮细胞生长因子（VEGF） ，再与 HRP 标记的检测抗体结合，形成抗体-
抗原-酶标抗体复合物，经过彻底洗涤后加底物 TMB 显色。TMB 在 HRP 酶的催化下转化成蓝
色，并在酸的作用下转化成最终的黄色。颜色的深浅和样品中的猪血管内皮细胞生长因子
（VEGF）呈正相关。用酶标仪在 450nm 波长下测定吸光度（OD值），通过标准曲线计算样品
中猪血管内皮细胞生长因子（VEGF）含量。
 
试剂盒组成：
试剂盒组成  48 孔配置  96 孔配置  保存
说明书  1份  1 份  
封板膜  2片  2 片  
密封袋  1个  1 个  
酶标包被板  1×48  1×96  2-8℃保存
标准品  0.3ml×6 管  0.3ml×6 管  2-8℃保存
酶标试剂  5 ml×1瓶  10 ml×1 瓶  2-8℃保存
样品稀释液  3 ml×1瓶  6 ml×1 瓶  2-8℃保存
显色剂 A 液  3 ml×1瓶  6 ml×1 瓶  2-8℃保存
显色剂 B 液  3 ml×1瓶  6 ml×1 瓶  2-8℃保存
终止液  3 ml×1瓶  6 ml×1 瓶  2-8℃保存
20×浓缩洗涤液  15ml×1瓶  25ml×1 瓶  2-8℃保存
注：标准品浓度依次为：240、120、60、30、15、0 pg/mL.
 
样本处理及要求：
1. 血清：室温血液自然凝固 10-20分钟，离心20 分钟左右（2000-3000转/分）。仔细收集
上清，保存过程中如出现沉淀，应再次离心。
2. 血浆：应根据标本的要求选择 EDTA 或柠檬酸钠作为抗凝剂，混合 10-20 分钟后，离心
20分钟左右（2000-3000 转/分）。仔细收集上清，保存过程中如有沉淀形成，应该再次
离心。
3. 尿液：用无菌管收集，离心20分钟左右（2000-3000 转/分）。仔细收集上清，保存过程
中如有沉淀形成，应再次离心。胸腹水、脑脊液参照实行。
4. 细胞培养上清：检测分泌性的成份时，用无菌管收集。离心 20 分钟左右（2000-3000转 
/分）。仔细收集上清。检测细胞内的成份时，用 PBS（PH7.2-7.4）稀释细胞悬液，细胞
浓度达到 100 万/ml左右。通过反复冻融，以使细胞破坏并放出细胞内成份。离心 20 分
钟左右（2000-3000转/分）。仔细收集上清。保存过程中如有沉淀形成，应再次离心。
5. 组织标本：切割标本后，称取重量。加入一定量的 PBS，PH7.4。用液氮迅速冷冻保存备
用。标本融化后仍然保持 2-8℃的温度。加入一定量的 PBS（PH7.4），用手工或匀浆器将
标本匀浆充分。离心 20 分钟左右（2000-3000 转/分）。仔细收集上清。分装后一份待检
测，其余冷冻备用。
6. 标本采集后尽早进行提取，提取按相关文献进行，提取后应尽快进行实验。若不能马上
进行试验，可将标本放于-20℃保存，但应避免反复冻融.
7. 不能检测含 NaN3的样品，因 NaN3抑制辣根过氧化物酶的（HRP）活性。
 
操作步骤
1.  标准品的加样：设置标准品孔和样本孔，标准品孔各加不同浓度的标准品50μ L； 。
2.  加样：分别设空白孔（空白对照孔不加样品及酶标试剂，其余各步操作相同）、待测样
品孔。在酶标包被板上待测样品孔中先加样品稀释液 40μ l，然后再加待测样品 10μ l
（样品最终稀释度为 5 倍）。加样将样品加于酶标板孔底部，尽量不触及孔壁，轻轻晃
动混匀。
3.  加酶：每孔加入酶标试剂 100μ l，空白孔除外。
4.  温育：用封板膜封板后置 37℃温育 60分钟。
5.  配液：将 20倍浓缩洗涤液用蒸馏水 20倍稀释后备用。
6.  洗涤：小心揭掉封板膜，弃去液体，甩干，每孔加满洗涤液，静置 30 秒后弃去，如此
重复5 次，拍干。
7.  显色：每孔先加入显色剂 A50μ l，再加入显色剂 B50μ l，轻轻震荡混匀，37℃避光显
色 15 分钟. 
8.  终止：每孔加终止液50μ l，终止反应（此时蓝色立转黄色） 。
9.  测定：以空白孔调零，450nm波长依序测量各孔的吸光度（OD 值） 。 测定应在加终止液
后 15 分钟以内进行。
 
注意事项：
1． 试剂盒从冷藏环境中取出应在室温平衡15-30分钟后方可使用，酶标包被板开封后如未
用完，板条应装入密封袋中保存。
2． 浓洗涤液可能会有结晶析出，稀释时可在水浴中加温助溶，洗涤时不影响结果。
3． 各步加样均应使用加样器，并经常校对其准确性，以避免试验误差。一次加样时间最好
控制在5 分钟内，如标本数量多，推荐使用排枪加样。
4． 请每次测定的同时做标准曲线，最好做复孔。如标本中待测物质含量过高（样本 OD 值
大于标准品孔第一孔的 OD值），请先用样品稀释液稀释一定倍数（n 倍）后再测定，计
算时请最后乘以总稀释倍数（×n×5）。
5． 封板膜只限一次性使用，以避免交叉污染。
6． 底物请避光保存。
7． 严格按照说明书的操作进行，试验结果判定必须以酶标仪读数为准.
8． 所有样品，洗涤液和各种废弃物都应按传染物处理。
9． 本试剂不同批号组分不得混用。
10. 如与英文说明书有异，以英文说明书为准。 
 
 
 
计算：
以标准物的浓度为横坐标，OD值为纵坐标，    
在坐标纸上绘出标准曲线，根据样品的OD      
值由标准曲线查出相应的浓度；再乘以稀释      
倍数；或用标准物的浓度与 OD值计算出标      
准曲线的直线回归方程式，将样品的 OD值      
代入方程式，计算出样品浓度，再乘以稀释      
倍数，即为样品的实际浓度。                   
 
                                               

 
 
 
试剂盒性能：
1.样品线性回归与预期浓度相关系数 R值为 0.95以上。
2.批内变异系数与批间变异系数应分别小于 10%和15% 。
 
检测范围：                                              
7.5 pg/mL - 240 pg/mL
 
灵敏度：                                              
最低检测浓度小于 1.0 pg/mL
 
保存条件及有效期：
1.试剂盒保存： 2-8℃。
2．有效期： 6个月 
  
 
Procine Vascular Endothelial cell Growth
Factor
FOR RESEARCH USE ONLY
 
Drug Names
Generic Name：Procine Vascular Endothelial cell Growth Factor
(VEGF) ELISA Kit.
Purpose
This kit allows for the determination of VEGF concentrations
in  Procine  serum, plasma, tissue homogenates and other
biological fluids.
Principle of the assay
The kit assay Procine VEGF level in the sample, use Purified
Procine  VEGF  antibody to coat  microtiter plate wells, make
solid-phase antibody,  then add VEGF  to  the wells, Combined
antibody which With HRP  labeled,  become
antibody-antigen-enzyme-antibody complex, after  washing
Completely, Add TMB substrate solution,TMB substrate becomes
blue color At HRP enzyme-catalyzed, reaction is terminated by
the addition of a sulphuric acid solution and the color change is
measured spectrophotometrically at a wavelength of 450 nm. The
concentration of  VEGF  in the samples is then determined by  
 
comparing the O.D. of the samples to the standard curve.  
 
Materials provided with the kit
Materials
provided with
the kit
48determinations
96
determinations
Storage
User manual  1  1  
Closure plate
membrane
2  2  
Sealed bags  1  1  
Microelisa
stripplate
1  1  2-8℃
Standard  0.3ml×6 bottle  0.3ml×6 bottle  2-8℃
HRP-Conjugate
reagent
5ml×1 bottle  10ml×1 bottle  2-8℃
Sample diluent  3ml×1 bottle  6ml×1 bottle  2-8℃
Chromogen
Solution A
3ml×1 bottle  6ml×1 bottle  2-8℃
Chromogen
Solution B
3ml×1 bottle  6ml×1 bottle  2-8℃
Stop Solution  3ml×1 bottle  6ml×1 bottle  2-8℃
20×Wash
solution
15ml×1 bottle  25ml×1 bottle  2-8℃
Note: Standard concentration was followed by:    
 
240、120、60、30、15、0 pg/mL.
Specimen requirements
1.  serum-  coagulation at room  temperature 10-20 mins ，
centrifugation 20-min  at the speed of  2000-3000 r.p.m.
remove  supernatant,  If precipitation appeared,  Centrifugal
again.
2.  plasma-use suited EDTA  or  citrate  plasma  as an
anticoagulant,mix 10-20 mins ,centrifugation 20-min at the
speed  of  2000-3000 r.p.m. remove  supernatant,  If
precipitation appeared, Centrifugal again.
3.  Urine-collect sue a sterile container,  centrifugation 20-min at
the speed of  2000-3000 r.p.m. remove  supernatant,  If
precipitation appeared, Centrifugal again. The Operation of
Hydrothorax and cerebrospinal fluid Reference to it.
4.  cell culture supernatant-detect secretory components, collect
sue a sterile container, centrifugation 20-min at the speed of
2000-3000 r.p.m. remove supernatant,detect the composition
of cells, Dilut cell suspension with PBS（PH7.2-7.4）, Cell
concentration reached 1 million / ml, repeated freeze-thaw
cycles, damage cells and release of intracellular components,
centrifugation 20-min  at the speed of  2000-3000 r.p.m.
remove  supernatant,  If precipitation appeared,  Centrifugal  
 
again.
5.  Tissue samples- After cutting samples, check the weight,add
PBS（PH7.2-7.4）,  Rapidly frozen with liquid nitrogen,
maintain samples at 2-8℃ after melting,add PBS（PH7.4）,
Homogenized by hand or Grinders, centrifugation 20-min at
the speed of 2000-3000 r.p.m. remove supernatant.
6.  extract  as soon as possible after Specimen collection,and
according to the relevant literature, and should be experiment
as soon as possible after the extraction. If it can’t, specimen can
be kept in  -20 ℃  to preserve, Avoid repeated freeze-thaw
cycles.
7.  Can’t detect the sample which contain NaN3, because NaN3
inhibits HRP active.
Assay procedure
1. Add standard: Set Standard wells, testing sample wells. Add
standard 50μl to standard well.
2.add sample： Set blank wells separately (blank comparison wells
don’t add sample and HRP-Conjugate reagent, other each step
operation is same). testing sample well. add Sample dilution
40μl to testing sample well, then add  testing sample 10μl
(sample final dilution  is 5-fold), add sample to wells  , don’t
touch the well wall as far as possible, and Gently mix.  
 
3.add enzyme：Add HRP-Conjugate reagent 100μl to each well,
except  blank well.  
4.Incubate: After closing plate with  Closure plate
membrane ,incubate for 60 min at 37℃.
5.Configurate liquid:  20-fold  wash solution diluted  20-fold
with distilled water and reserve.
6.washing：Uncover Closure plate membrane, discard Liquid, dry
by swing, add washing buffer to every well, still for 30s then
drain, repeat 5 times, dry by pat.
7.color ： Add  Chromogen Solution A  50ul and  Chromogen
Solution B to each well, evade the light preservation for 15 min
at 37℃
8.Stop the reaction：Add Stop Solution 50μl  to each well, Stop the
reaction(the blue color change to yellow color).
9.assay： take blank well as zero , Read absorbance at 450nm after
Adding Stop Solution and within 15min.  
 
Important notes
1.  The kit takes out from the refrigeration environment should be
balanced 15-30 minutes in the room temperature,  ELISA
plates coated if has not use up after opened, the plate should be
stored in Sealed bag.
2.  washing buffer will Crystallization separation, it can be
heated the water helps dissolve when dilute . Washing does not
affect the result.
3.  add Sample with sampler Each step, And proofread its
accuracy frequently, avoids the experimental error. add
sample within 5 mins, if the number of sample is much ,
recommend to use Volley .
4.  if the testing material content is excessively higher  (The
sample OD is bigger than the first standard well ),please dilute
Sample (n-fold), Please diluente  and  multiplied by the
dilution factor.（×n×5）.
5.  Closure plate membrane  only limits the disposable use, to
avoid cross-contamination.
6.  The substrate evade the light preservation.
7.  Please according to use instruction strictly, The test result
determination must take the  microtiter plate reader  as a
standard.  

8.  All samples, washing buffer and each kind of reject should
according to infective material process.
9.  Do not mix reagents with those from other lots.  
 
Calculate
 
 
   
                                                     This chartis for reference only
 
 
 
 
 
 
Assay range
7.5 pg/mL - 240 pg/mL
 
Sensitivity
The minimum detectable dose is typically less than 1.0 pg/mL
 
Storage and validity
1．Storage：  2-8℃.
2．validity： six months.
Take  the standard density as the
horizontal, the  OD value for the
vertical  ,draw the standard curve on
graph paper, Find out the corresponding
density according to the sample OD value
by the Sample curve, multiplied by the
dilution multiple, or calculate the
straight line regression equation of  the
standard curve with the standard density
and the OD value ,with the sample OD
value in the equation, calculate the
sample density,  multiplied by the
dilution  factor, the result is the sample
actual density.