小鼠血管内皮生长因子 （VEGF）酶联免疫分析试剂盒使用说明书
本试剂盒仅供研究使用。
检测范围： 96T
15ng/L -350ng/L
使用目的：
本试剂盒用于测定小鼠血清、血浆及相关液体样本中血管内皮生长因子 （VEGF）含量。
实验原理
本试剂盒应用双抗体夹心法测定标本中小鼠血管内皮生长因子 （VEGF）水平。用纯化的
小鼠血管内皮生长因子 （VEGF）抗体包被微孔板，制成固相抗体，往包被单抗的微孔中依次
加入血管内皮生长因子 （VEGF），再与HRP 标记的血管内皮生长因子 （VEGF）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*终的黄色。颜色的深浅和样品中的血管内皮生长因
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
样品中小鼠血管内皮生长因子 （VEGF）浓度。
试剂盒组成
1 30 倍浓缩洗涤液 20ml×1 瓶 7 终止液 6ml×1 瓶
2 酶标试剂 6ml×1 瓶 8 标准品（640ng/L） 0.5ml×1 瓶
3 酶标包被板 12 孔×8 条 9 标准品稀释液 1.5ml×1 瓶
4 样品稀释液 6ml×1 瓶 10 说明书 1 份
5 显色剂A 液 6ml×1 瓶 11 封板膜 2 张
6 显色剂B 液 6ml×1/瓶 12 密封袋 1 个
标本要求
1．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
2．不能检测含NaN3 的样品，因NaN3 抑制辣根过氧化物酶的（HRP）活性。
小鼠血管内皮生长因子 （VEGF）酶联免疫分析试剂盒
操作步骤
1. 标准品的稀释：本试剂盒提供原倍标准品一支，用户可按照下列图表在小试管中进行稀
释。
320ng/L 5 号标准品 150μl 的原倍标准品加入150μl 标准品稀释液
160ng/L 4 号标准品 150μl 的5 号标准品加入150μl 标准品稀释液
80ng/L 3 号标准品 150μl 的4 号标准品加入150μl 标准品稀释液
40ng/L 2 号标准品 150μl 的3 号标准品加入150μl 标准品稀释液
20ng/L 1 号标准品 150μl 的2 号标准品加入150μl 标准品稀释液
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）、标准孔、
待测样品孔。在酶标包被板上标准品准确加样50μl，待测样品孔中先加样品稀释液40μl，
然后再加待测样品10μl（样品*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. 温育：用封板膜封板后置37℃温育30 分钟。
4. 配液：将30 倍浓缩洗涤液用蒸馏水30 倍稀释后备用
5. 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。
6. 加酶：每孔加入酶标试剂50μl，空白孔除外。
7. 温育：操作同3。
8. 洗涤：操作同5。
9. 显色：每孔先加入显色剂A50μl，再加入显色剂B50μl，轻轻震荡混匀，37℃避光显色
15 分钟.
10. 终止：每孔加终止液50μl，终止反应（此时蓝色立转黄色）。
11. 测定：以空白空调零，450nm 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。
操作程序总结：
小鼠血管内皮生长因子 （VEGF）酶联免疫分析试剂盒计算
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。
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
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，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*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。
保存条件及有效期
1．试剂盒保存：；2-8℃。
2．有效期：6 个月
Mouse VEGF
FOR RESEARCH USE ONLY
Assay range：15ng/L -350ng/L 96 determinations
Purpose
This kit allows for the determination of VEGF concentrations in Mouse serum, cell
culture supernates ａnd other biological fluids
Principle of the assay
The kit assay Mouse VEGF level in the sample，use Purified Mouse VEGF antibody to
coat microtiter plate wells, make solid-phase antibody, then add VEGF to wells, Combined
VEGF 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 Mouse VEGF in the samples is then determined by comparing the O.D. of
the samples to the standard curve.
Materials provided with the kit
1 wash solution 20ml×1bottle 7 Stopp Solution 6ml×1 bottle
2 HRP-Conjugate reagent 6ml×1 bottle 8 Standard（640ng/L） 0.5ml×1 bottle
3 Microelisa stripplate 12well×8strips 9 Standard diluent 1.5ml×1bottle
4 Sample diluent 6ml×1 bottle 10 Instruction 1
5 Chromogen Solution A 6ml×1 bottle 11
Closure plate
membrane
2
6 Chromogen Solution B 6ml×1 bottle 12 Sealed bags 1
Specimen requirements
1. extract as soon as possible after Specimen collection,and according to the relevant
RD
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.
2. Can’t detect the sample which contain NaN3, because NaN3 inhibits HRP active.
Assay procedure
1. Dilute ａnd add sample:Dilute Original density Standard as follow table:
320ng/L 5 Standard 150μl Original density Standard+150μl Standard diluent
160ng/L 4 Standard 150μl 5 Standard+150μl Standard diluent
80ng/L 3 Standard 150μl 4 Standard+150μl Standard diluent
40ng/L 2 Standard 150μl 3 Standard +150μl Standard diluent
20ng/L 1 Standard 150μl 2 Standard +150μl Standard diluent
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.Incubate: After closing plate with Closure plate membrane ,incubate for 30 min at 37℃.
4.Configurate liquid: 30-fold（or 20-fold） wash solution diluted 30-fold （or 20-fold） with distilled
water ａnd reserve.
5.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.
6.add enzyme：Add HRP-Conjugate reagent 50μl to each well, except blank well.
7.incubate：Operation with 3.
8.washing：Operation with 5.
9.color：Add Chromogen Solution A 50ul ａnd Chromogen Solution B to each well, evade the
light preservation for 15 min at 37℃
10.Stop the reaction：Add Stop Solution50μl to each well, Stop the reaction（the blue color
change to yellow color）.
11.assay：take blank well as zero , Read absorbance at 450nm after Adding Stop Solution and
within 15min.
Steps description
Standard, Sample diluent
Add Standard, Sample diluent, incubate for 30 min at 37℃.
Wash 5 time,Add HRP-Conjugate reagent, incubate for 30 min at 37℃.
Wash 5 times,Add Chromogen Solution A ａnd B, incubate for 30 min at 37℃.
Add Stopp Solution
Read absorbance at 450nm within 15 min
calculate
Calculate
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.
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 min, 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.
Storage ａnd validity
1．Storage： 2-8℃.
小鼠血管内皮生长因子 （VEGF）酶联免疫分析试剂盒
2．validity： six months
电议 型 号：
