活性氧簇ELISA试剂盒，大鼠ROS试剂盒说明书
检测原理
试剂盒采用双抗体一步夹心法酶联免疫吸附试验（ELISA）。往预先包被活性氧簇（ROS）抗体的包被微孔中，依次加入标本、标准品、HRP标记的检测抗体，经过温育并*洗涤。用底物TMB显色，TMB在过氧化物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活性氧簇（ROS）呈正相关。用酶标仪在450nm 波长下测定吸光度（OD 值），计算样品浓度。
样品收集、处理及保存方法
1. 血清：使用不含热原和内毒素的试管，操作过程中避免任何细胞刺激，收集血液后，3000 转离心10 分钟将血清和红细胞迅速小心地分离。
2. 血浆：EDTA、柠檬酸盐或肝素抗凝。3000 转离心30 分钟取上清。
3. 细胞上清液：3000 转离心10 分钟去除颗粒和聚合物。
4. 组织匀浆：将组织加入适量生理盐水捣碎。3000 转离心10 分钟取上清。
5. 保存：如果样本收集后不及时检测，请按一次用量分装，冻存于-20℃，避免反复冻融，在室温下解冻并确保样品均匀地充分解冻。
自备物品
1. 酶标仪（450nm）
2. 高精度加样器及枪头：0.5-10uL、2-20uL、20-200uL、200-1000uL
3. 37℃恒温箱
操作注意事项
1. 试剂盒保存在2-8℃，使用前室温平衡20 分钟。从冰箱取出的浓缩洗涤液会有结晶，这属于正常现象，水浴加热使结晶*溶解后再使用。
2. 实验中不用的板条应立即放回自封袋中，密封（低温干燥）保存。
3. 严格按照说明书中标明的时间、加液量及顺序进行温育操作。4. 所有液体组分使用前充分摇匀。
试剂盒组成
名称96孔配置48孔配置备注
微孔酶标板12 孔×8 条12 孔×4 条无
标准品（480 U/ml） 0.5mL 0.5mL 无
标准品稀释液6mL 3mL 无
样本稀释液6mL 3mL 无
检测抗体-HRP 10mL 5mL 无
20×洗涤缓冲液25mL 15mL 按说明书进行稀释
底物A 6mL 3mL 无
底物B 6mL 3mL 无
终止液6mL 3mL 无
封板膜2 张2 张无
说明书1 份1 份无
自封袋1 个1 个无
注：标准品用标准品稀释液倍比稀释为：480、240、120、60、30、15 U/ml
试剂的准备
20×洗涤缓冲液的稀释：蒸馏水按1：20 稀释，即1 份的20×洗涤缓
冲液加19 份的蒸馏水。
洗板方法
1. 手工洗板：甩尽孔内液体，每孔加满洗涤液，静置1min 后甩尽孔内液体，在吸水纸上拍干，如此洗板5 次。
2. 自动洗板机：每孔注入洗液350μL，浸泡1min，洗板5 次。
操作步骤
1. 从室温平衡20min 后的铝箔袋中取出所需板条，剩余板条用自封袋密封放回4℃。
2. 设置标准品孔和样本孔。标准品孔各加不同浓度的标准品50μL；待测样本孔先加样本10μL，再加样本稀释液40μL；空白孔不加。
3. 除空白孔外，标准品孔和样本孔中每孔加入辣根过氧化物酶（HRP）标记的检测抗体100μL，用封板膜封住反应孔，37℃水浴锅或恒温箱温育60min。
4. 弃去液体，吸水纸上拍干，每孔加满洗涤液，静置1min，甩去洗涤液，吸水纸上拍干，如此重复洗板5 次（也可用洗板机洗板）。
5. 每孔加入底物A、B 各50μL，37℃避光孵育15min。
6. 每孔加入终止液50μL，15min 内，在450nm 波长处测定各孔的OD 值。
结果判断
绘制标准曲线：在Excel 工作表中，以标准品浓度作横坐标，对应OD 值作纵坐标，绘制出标准品线性回归曲线，按曲线方程计算各样
本浓度值。
试剂盒性能
1. 准确性：标准品线性回归与预期浓度相关系数R 值，大于等于0.9900。
2. 灵敏度：zui低检测浓度小于1.0 U/ml。
3. 特异性：不与其它可溶性结构类似物交叉反应。
4. 重复性：板内、板间变异系数均小于15%。
5. 贮藏：2-8℃，避光防潮保存。
6. 有效期：6 个月
活性氧簇ELISA试剂盒，大鼠ROS试剂盒说明书
instruction
Intended use
This ROS ELISA kit is intended Laboratory for Research use only and is not for
use in diagnostic or therapeutic procedures.The Stop Solution changes the color
from blue to yellow and the intensity of the color is measured at 450 nm using a
spectrophotometer. In order to measure the concentration of ROS in the sample,
this ROS ELISA Kit includes a set of calibration standards. The calibration
standards are assayed at the same time as the samples and allow the operator to
produce a standard curve of Optical Density versus ROS concentration. The
concentration of ROS in the samples is then determined by comparing the O.D. of
the samples to the standard curve.
Sample collection and storages
Serum - Use a serum separator tube and allow samples to clot for 30 minutes
before centrifugation for 10 minutes at approximay 3000×g. Remove serum and
assay immediay or aliquot and store samples at -20℃ or -80℃.Avoid repeated
freeze-thaw cycles
Plasma - Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge
samples for 30 minutes at 3000×g at 2-8℃ within 30 minutes of collection. Store
samples at -20℃or -80℃. Avoid repeated freeze-thaw cycles.
Cell culture supernates and other biological fluids - Remove particulates
by centrifugation and assay immediay or aliquot and store samples at -20℃or
-80℃. Avoid repeated freeze-thaw cycles.
Note: The samples shoule be centrifugated dequay and no hemolysis or
granule was allowed.
Materials required but not supplied
1. Standard microplate reader（450nm）
2. Precision pipettes and Disposable pipette tips.
3. 37 ℃ incubator
Precautions
1. Do not substitute reagents from one kit to another. Standard, conjugate and
microplates are matched for optimal performance. Use only the reagents supplied by
manufacturer.
2. Do not remove microplate from the storage bag until needed. Unused strips
should be stored at 2-8°C in their pouch with the desiccant provided.
3. Mix all reagents before using.Remove all kit reagents from refrigerator and allow them to reach room temperature
（ 20-25°C）
Materials supplied
Name 96 determinations 48 determinations
Microelisa stripplate 12*8strips 12*4strips
Standard （480 U/ml） 0.5ml 0.5ml
Standard Diluent 6.0ml 3.0ml
Sample Diluent 6.0ml 3.0ml
HRP-Conjugate reagent 10.0ml 5.0ml
20X Wash solution 25ml 15ml
Chromogen Solution A 6.0ml 3.0ml
Chromogen Solution B 6.0ml 3.0ml
Stop Solution 6.0ml 3.0ml
Closure plate membrane 2 2
User manual 1 1
Sealed bags 1 1
Note: Standard concentration was followed by:480,240,120,60,30,15 U/ml
Reagent preparation
20×wash solution:Dilute with Distilled or deionized water 1:20.
Assay procedure
1. Prepare all r e a g e n t s before starting assay procedure. It is recommended that
all Standards and Samples be added in duplicate to the Microelisa Stripplate.
2. Add standard: Set Standard wells, testing sample wells. Add standard 50μl to
standard well.
3. Add Sample: Add Sample 10μl Then add Sample Diluent 40μl to testing
sample well; Blank well doesn’t add anyting.
4. Add 100μl of HRP-conjugate reagent to each well except blank well, cover
with an adhesive strip and incubate for 60 minutes at 37°C.
4. Aspirate each well and wash, repeating the process four times for a total of five
washes. Wash by filling each well with Wash Solution （400μl） using a squirt bottle,
manifold dispenser or autowasher. Complete removal of liquid at each step is
essential to good performance. After the last wash, remove any remaining Wash
Solution by aspirating or decanting. Invert the plate and blot it against clean paper
towels.
5. Add chromogen solution A 50μl and chromogen solution B 50μl to each well.
Gently mix and incubate for 15 minutes at 37°C. Protect from light.
6. Add 50μl Stop Solution to each well. The color in the wells should change
from blue to yellow. If the color in the wells is green or the color change does not
appear uniform, gently tap the plate to ensure thorough mixing.
7. Read the Optical Density （O.D.） at 450 nm using a microtiter plate reader
within 15 minutes.
Calculation of results
1. This standard curve is used to determine the amount in an unknown sample.The standard curve is generated by plotting the average O.D. （450 nm）
obtained for each of the six standard concentrations on the vertical （Y） axis
versus the corresponding concentration on the horizontal （X） axis.
2. First, calculate the mean O.D. value for each standard and sample. All O.D.
values, are subtracted by the mean value of the zero standard before result
interpretation. Construct the standard curve using graph paper or statistical
software.
3. To determine the amount in each sample, first locate the O.D. value on the
Y-axis and extend a horizontal line to the standard curve. At the point of
intersection, draw a vertical line to the X-axis and read the corresponding
concentration.
4. Any variation in operator, pipetting and washing technique, incubation time or
temperature, and kit age can cause variation in result. Each user should obtain
their own standard curve.
5. The sensitivity by this assay is 1.0 U/ml
6. Standard curve
Storage： 2-8℃.
validity： six months.
