植物过氧化氢酶（CAT）ELISA试剂盒操作说明

【资料简介】

1
植物过氧化氢酶（CAT）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：本试剂盒用于测定植物组织，细胞及相
关液体样本中过氧化氢酶（CAT）的含量。
实验原理：
本试剂盒应用双抗体夹心法测定标本中过氧化氢酶（CAT）水平。用纯化的过氧化氢酶
（CAT） 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过氧化氢酶 （CAT） ，
再与 HRP 标记的过氧化氢酶（CAT）抗体结合，形成抗体-抗原-酶标抗体复合物，经过*
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zui
终的黄色。颜色的深浅和样品中的过氧化氢酶（CAT）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过氧化氢酶（CAT）浓度。
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（48） 2片（96）
密封袋 1 个 1个
酶标包被板 1×48 1×96 2-8℃保存
标准品：13.5ng/ml 0.5ml×1 瓶 0.5ml×1瓶 2-8℃保存
标准品稀释液 1.5ml×1 瓶 1.5ml×1瓶 2-8℃保存
酶标试剂 3 ml×1 瓶 6 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 3ml×1 瓶 6ml×1 瓶 2-8℃保存
浓缩洗涤液 （20ml×20倍）×1瓶 （20ml×30 倍）×1瓶 2-8℃保存
样本处理及要求：
1. 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。
2. 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。
3. 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.
4. 不能检测含 NaN3的样品，因 NaN3 抑制辣根过氧化物酶的（HRP）活性。2
操作步骤
1. 标准品的稀释与加样：在酶标包被板上设标准品孔 10孔，在*、第二孔中分别加标
准品 100μl，然后在*、第二孔中加标准品稀释液 50μl，混匀；然后从*孔、第二
孔中各取 100μl 分别加到第三孔和第四孔， 再在第三、 第四孔分别加标准品稀释液 50μl，
混匀；然后在第三孔和第四孔中先各取 50μl 弃掉，再各取 50μl 分别加到第五、第六孔
中，再在第五、第六孔中分别加标准品稀释液 50ul，混匀；混匀后从第五、第六孔中各
取 50μl 分别加到第七、第八孔中，再在第七、第八孔中分别加标准品稀释液 50μl，混
匀后从第七、第八孔中分别取 50μl 加到第九、第十孔中，再在第九第十孔分别加标准
品稀释液 50μl，混匀后从第九第十孔中各取 50μl 弃掉。 （稀释后各孔加样量都为 50μl，
浓度分别为 9 ng/ml，6 ng/ml，3 ng/ml，1.5 ng/ml， 0.75 ng/ml） 。
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 40μl，然后再加待测样品 10μl（样
品zui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（48T 的 20倍）倍浓缩洗涤液用蒸馏水 30（48T 的 20 倍）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。
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，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zui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。3
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。
（此图仅供参考）
试剂盒性能：
1.样品线性回归与预期浓度相关系数 R 值为 0.92以上。
2.批内与批间应分别小于 9%和 15%
检测范围：
0.3ng/ml -10 ng/ml
保存条件及有效期：
1.试剂盒保存：2-8℃。
2．有效期：6 个月4
FOR RESEARCH USE ONLY
Plant Catalase
Drug Names
Generic Name：Plant Catalase(CAT) ELISA Kit.
Purpose
This kit allows for the determination of CAT in Plant tissue, cell culture
supernatant and other biological fluids.
Principle of the assay
The kit assay Plant CAT level in the sample，use Purified Plant CAT
antibody to coat microtiter plate wells, make solid-phase antibody, then add
CAT to wells, Combined CAT antibody which With HRP labeled ,become
antibody - antigen - enzyme-antibody complex, after washing Compley, 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 CAT in the samples is then
determined by comparing the O.D. of the samples to the standard curve.5
Materials provided with the kit
Materials provided
with the kit
48determinations 96 determinations
Stora
ge
User manual 1 1
Closure plate
membrane
2 2
Sealed bags 1 1
Microelisa stripplate 1 1 2-8℃
Standard：13.5ng/ml 0.5ml×1 bottle 0.5ml×1 bottle 2-8℃
Standard diluent 1.5ml×1 bottle 1.5ml×1 bottle 2-8℃
HRP-Conjugate
reagent
3ml×1 bottle 6ml×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℃
wash solution
（20ml×20 fold）
×1bottle
（20ml×30 fold）
×1bottle
2-8℃
Specimen requirements
1. 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.
2. 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
3. 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.
4. Can’t detect the sample which contain NaN3, because NaN3 inhibits HRP
active.
Assay procedure
1.Dilute and add sample to Standard: set 10 Standard wells on the ELISA
plates coated, add Standard 100μl to the first and the second well, then add
Standard dilution 50μl to the first and the second well, mix; take out 100μl
form the first and the second well then add it to the third and the forth well
separay. then add Standard dilution 50μl to the third and the forth
well ,mix ; then take out 50μl from the third and the forth well discard, add
50μl to the fifth and the sixth well ,then add Standard dilution 50μl to the fifth
and the sixth well, mix ; take out 50μl from the fifth and the sixth well and add
to the seventh and the eighth well, then add Standard dilution 50μl to the
seventh and the eighth well ,mix ; take out 50μl from the seventh and the
eighth well and add to the ninth and the tenth well, add Standard dilution 50μl
to the ninth and the tenth well, mix , take out 50μl from the ninth and the tenth
well discard(add Sample 50μl to each well after Diluting ,(density: 9 ng/ml，6
ng/ml，3 ng/ml，1.5 ng/ml， 0.75 ng/ml)
2.add sample：Set blank wells separay (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℃.7
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.
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.8
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
Assay range
0.3ng/ml -10 ng/ml
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
This chart for reference only9
Storage and validity
1．Storage： 2-8℃.
2．validity： six months.