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Rat Cortisol ELISA Kit
Catalog No. CSB-E05112r
（96 tests）
This immunoassay kit allows for the in vitro rapid detection of rat Cortisol
concentrations in serum, plasma.
Expiration date six months from the date of manufacture
FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES.
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INTRODUCTION
Cortisol is a corticosteroid hormone or glucocorticoid produced
by the adrenal cortex, which is part of the adrenal gland （in the
zona fasciculata and the zona reticularis of the adrenal cortex）. It
is usually referred to as the "stress hormone" as it is involved in
response to stress and anxiety, controlled by CRH. It increases
blood pressure and blood sugar, and reduces immune
responses. Various synthetic forms of cortisol are used to treat a
variety of different illnesses. The most well-known of these is a
natural metabolic intermediary of cortisol called hydrocortisone.
When first introduced as a treatment for rheumatoid arthritis,
hydrocortisone was referred to as Compound E.
PRINCIPLE OF THE ASSAY
This assay employs the competitive inhibition enzyme
immunoassay technique. An anti-antibody specific to the
antibody of cortisol has been pre-coated onto a microplate.
Standards or samples are added to the appropriate microtiter
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plate wells with an antibody specific for cortisol and Horseradish
Peroxidase （HRP） conjugated cortisol, then incubated. A
competitive inhibition reaction is launched between cortisol
（Standards or samples） and Horseradish Peroxidase （HRP）
conjugated cortisol with the antibody specific for cortisol. The
more the amount of cortisol in samples, the less antibody bound
by Horseradish Peroxidase （HRP） conjugated cortisol. The
substrate solutions are added to the wells, respectively. And the
color develops in opposite to the amount of cortisol in the sample.
The color development is stopped and the intensity of the color is
measured.
DETECTION RANGE
The standard curve concentrations used for the ELISA’s were
20ng/ml, 5ng/ml, 1.25ng/ml, 0.31ng/ml, 0.08ng/ml.
SPECIFICITY
This assay recognizes cortisol. No significant cross-reactivity or
interference was observed.
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MATERIALS PROVIDED
Reagent Quantity
Assay plate 1
Standards （S1-S5） 5
Antibody 1 x 6 ml
HRP-conjugate 1 x 6 ml
Wash Buffer
1 x 15 ml
20×concentrate）
Substrate A 1 x 7 ml
Substrate B 1 x 7 ml
Stop Solution 1 x 7 ml
Standard S1 S2 S3 S4 S5
Concentration（ng/ml） 0.08 0.31 1.25 5 20
STORAGE
1. Unopened test kits should be stored at 2-8°C upon receipt
and the microtiter plate should be kept in a sealed bag. The
test kit may be used throughout the expiration date of the kit,
provided it is stored as prescribed above. Refer to the
package label for the expiration date.
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2. Opened test plate should be stored at 2-8°C in the aluminum
foil bag with desiccants to minimize exposure to damp air. The
kits will remain stable until the expiring date shown, provided it
is stored as prescribed above.
3. A microtiter plate reader with a bandwidth of 10 nm or less
and an optical density range of 0-3 OD or greater at 450nm
wavelength is acceptable for use in absorbance
measurement.
TECHNICAL HINTS
1. Bring all reagents and plate to room temperature for at least
30 minutes before use. Unused wells need store at 2-8℃and
avoid sunlight.
2. Centrifuge vials before opening to collect contents.
3. Wash Buffer If crystals have formed in the concentrate,
warm to room temperature and mix gently until the crystals
have compley dissolved. Dilute 15 ml of Wash Buffer
Concentrate into deionized or distilled water to prepare 300 ml
of Wash Buffer.
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4. To avoid cross-contamination, change pipette tips between
additions of each standard level, between sample additions,
and between reagent additions. Also, use separate reservoirs
for each reagent.
5. When using an automated plate washer, adding a 30 second
soak period following the addition of wash buffer, and/or
rotating the plate 180 degrees between wash steps may
improve assay precision.
6. Substrate Solution should remain colorless or light blue until
added to the plate. Keep Substrate Solution protected from
light. Substrate Solution should change from colorless or light
blue to gradations of blue.
7. Stop Solution should be added to the plate in the same order
as the Substrate Solution. The color developed in the wells
will turn from blue to yellow upon addition of the Stop Solution.
Wells that are green in color indicate that the Stop Solution
has not mixed thoroughly with the Substrate Solution.
Precaution: The Stop Solution provided with this kit is an acid solution. Wear
eye, hand, face, and clothing protection when using this material.
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OTHER SUPPLIES REQUIRED
Microplate reader capable of measuring absorbance at 450
nm, with the correction wavelength set at 540 nm or 570 nm.
Pipettes and pipette tips.
Deionized or distilled water.
Squirt bottle, manifold dispenser, or automated microplate
washer.
An incubator which can provide stable incubation conditions
up to 37°C±0.5°C.
SAMPLE COLLECTION AND STORAGE
Serum Use a serum separator tube （SST） and allow
samples to clot for 30 minutes before centrifugation for 15
minutes at 1000 g. Remove serum and assay immediay or
aliquot and store samples at -20°C. Centrifuge the sample
again after thawing before the assay. Avoid repeated
freeze-thaw cycles.
Plasma Collect plasma using citrate, EDTA, or heparin as
an anticoagulant. Centrifuge for 15 minutes at 1000 g within
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30 minutes of collection. Assay immediay or aliquot and
store samples at -20°C. Centrifuge the sample again after
thawing before the assay. Avoid repeated freeze-thaw cycles.
Note: Grossly hemolyzed samples are not suitable for use in this assay.
ASSAY PROCEDURE
Bring all reagents and samples to room temperature before use. It is
recommended that all samples, standards, and controls be assayed in duplicate.
All the reagents should be added directly to the liquid level in the well. The
pipette should avoid contacting the inner wall of the well.
1. Set a Blank well without any solution. Add 50µl of Standard or
Sample per well. Standard need test in duplicate.
2. Add 50µl of HRP-conjugate to each well （not to Blank well）,
then 50µl antibody to each well. Mix well and then incubate
for 1 hour at 37°C.
3. Fill each well with Wash Buffer （about 250µl）, stay for 10
seconds and Spinning. Repeat the process for a total of three
washes. Complete removal of liquid at each step is essential
to good performance. After the last wash, remove any
remaining Wash Buffer by aspirating or decanting. Invert the
plate and blot it against clean paper towels.
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4. Add 50µl of Substrate A and Substrate B to each well, mix
well. Incubate for 15 minutes at 37°C. Keeping the plate away
from drafts and other temperature fluctuations in the dark.
5. Add 50µl of Stop Solution to each well when the first four
wells containing the highest concentration of standards
develop obvious blue color. If color change does not appear
uniform, gently tap the plate to ensure thorough mixing.
6. Determine the optical density of each well within 10 minutes,
using a microplate reader set to 450 nm.
CALCULATION OF RESULTS
Using the professional soft "Curve Exert 1.3" to make a standard curve is
recommended, which can be downloaded from our web.
Average the duplicate readings for each standard, Blank, and
sample and subtract the optical density of Blank. Create a
standard curve by reducing the data using computer software
capable of generating a four parameter logistic （4-PL） curve-fit.
As an alternative, construct a standard curve by plotting the
mean absorbance for each standard on the y-axis against the
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concentration on the x-axis and draw a best fit curve through the
points on the graph. The data may be linearized by plotting the
log of the cortisol concentrations versus the log of the O.D. and
the best fit line can be determined by regression analysis. This
procedure will produce an adequate but less precise fit of the
data. If samples have been diluted, the concentration read from
the standard curve must be multiplied by the dilution factor.
LIMITATIONS OF THE PROCEDURE
The kit should not be used beyond the expiration date on the
kit label.
Do not mix or substitute reagents with those from other lots or
sources.
If samples generate values higher than the highest standard,
dilute the samples with the appropriate Diluent and repeat the
assay.
Any variation in operator, pipetting technique, washing
technique, incubation time or temperature, and kit age can
cause variation in binding.
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This assay is designed to eliminate interference by soluble
receptors, binding proteins, and other factors present in
biological samples. Until all factors have been tested in the
Quantikine Immunoassay, the possibility of interference
cannot be excluded.
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大鼠皮质醇 大鼠皮质醇 大鼠皮质醇 大鼠皮质醇（Cortisol） （Cortisol） （Cortisol） （Cortisol）ELISA ELISA ELISA ELISA 快速检测试剂盒 快速检测试剂盒 快速检测试剂盒 快速检测试剂盒
使用说明书 使用说明书 使用说明书 使用说明书
本试剂盒仅供研究使用 本试剂盒仅供研究使用 本试剂盒仅供研究使用 本试剂盒仅供研究使用
产品编号 产品编号 产品编号 产品编号：CSB-E05112r
检测范围 检测范围 检测范围 检测范围： ：： ：0.08 ng/ml -20 ng/ml
zui低检测限：0.05 ng/ml
特异性 特异性 特异性 特异性： ：： ：本试剂盒可同时检测天然或重组的皮质醇，且与其他相关蛋白基本
无交叉反应。
有效期 有效期 有效期 有效期：6 个月（2-8℃避光保存）
预期应用 预期应用 预期应用 预期应用： ：： ：ELISA法定量测定大鼠血清，血浆中皮质醇含量。
说明 说明 说明 说明
1. 浓洗涤液低温保存会有盐析出，稀释时可在水浴中加温助溶。
2. 刚开启的酶联板孔中可能会含有少许水样物质，此为正常现象，不会对实
验结果造成任何影响。
实验原理 实验原理 实验原理 实验原理
采用竞争酶联免疫法法检测皮质醇含量。首先用羊抗兔包被微孔板，制
备成固相二抗，然后加入待测标本及辣根过氧化物酶标记的皮质醇和抗皮质
醇抗体，使之形成包被二抗-抗皮质醇抗体-酶标记皮质醇的复合物。经显色后
在酶标仪测定吸光值（OD值），通过计算机或作图拟合浓度-吸光度曲线，反
算出待测标本中皮质醇含量。
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试剂盒组成及试剂配制 试剂盒组成及试剂配制 试剂盒组成及试剂配制 试剂盒组成及试剂配制
1. 酶 酶酶 酶联 联联 联板 板板 板（Assay plate ）： 一块（96
孔）。
2. 标准品 标准品 标准品 标准品（Standard）： 5×0.5ml/
瓶。
标准品 S1 S 2 S3 S4 S5
浓度
（ng/ml）
0.08 0.31 1.25 5 20
3. 酶结合物 酶结合物 酶结合物 酶结合物（ （（ （HRP-conjugate） ）） ）： 1×6ml/
瓶。
4. 抗体 抗体 抗体 抗体（ （（ （Antibody） ）） ）： 1×6ml/
瓶
5. 显色剂 显色剂 显色剂 显色剂 A（ （（ （Substrate A） ）） ）： 1×7ml/
瓶。
6. 显色剂 显色剂 显色剂 显色剂 B（ （（ （Substrate B） ）） ）： 1×7ml/
瓶。
7. 浓洗涤液 浓洗涤液 浓洗涤液 浓洗涤液（ （（ （Wash Buffer） ）） ）： 1×15ml/瓶，使用时每瓶用蒸馏水稀释 20
倍。
8. 终止液 终止液 终止液 终止液（ （（ （Stop Solution） ）） ）： 1×7ml/
瓶
需要而未提供的试剂和器材 需要而未提供的试剂和器材 需要而未提供的试剂和器材 需要而未提供的试剂和器材
1. 标准规格酶标仪
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2. 高速离心机
3. 电热恒温培养箱
4. 干净的试管和 Eppendof管
5. 系列可调节移液器及吸头，一次检测样品较多时，用多通道移液器
6. 蒸馏水，容量瓶等
标本的采集及保存 标本的采集及保存 标本的采集及保存 标本的采集及保存
1. 血清：全血标本请于室温放置 2 小时或 4℃过夜后于 1000 x g离心 20 分
钟，取上清即可检测，或将标本放于-20℃或-80℃保存，但应避免反复冻
融。
2. 血浆：可用 EDTA 或肝素作为抗凝剂，标本采集后 30 分钟内于 2 - 8° C
1000 x g 离心 15 分钟，或将标本放于-20℃或-80℃保存，但应避免反复
冻融。
注 注注 注： ：： ：以上标本置 以上标本置 以上标本置 以上标本置 4℃ ℃℃ ℃保存应小于 保存应小于 保存应小于 保存应小于 1 周 周周 周， ，， ，-20℃ ℃℃ ℃或 或或 或-80℃ ℃℃ ℃均应密封保存 均应密封保存 均应密封保存 均应密封保存， ，， ，-20℃ ℃℃ ℃不 不不 不
应超过 应超过 应超过 应超过 1 个月 个月 个月 个月， ，， ，-80℃ ℃℃ ℃不 不不 不应超过 应超过 应超过 应超过 2 个月 个月 个月 个月； ；； ；标本溶血会影响zui后检测结果 标本溶血会影响zui后检测结果 标本溶血会影响zui后检测结果 标本溶血会影响zui后检测结果， ，， ，因此 因此 因此 因此
溶血标本不宜进行此项检测 溶血标本不宜进行此项检测 溶血标本不宜进行此项检测 溶血标本不宜进行此项检测。 。。 。
操作步骤 操作步骤 操作步骤 操作步骤
1. 将各种试剂至室温〔18-25℃〕平衡半小时，取浓缩洗涤液，根据当批检
测数量，用蒸馏水上 1：20 稀释，混匀后备用。
2. 将酶标板取出，设一个空白对照孔、不加任何液体；每个标准点依次各设
两孔，每孔加入相应标准品 50ul；其余每个检测孔直接加待测标本 50ul。
3. 每孔加入酶结合物 50ul（空白对照孔除外），然后再各加入 50ul 抗体，充
分混匀，贴上不干胶封片，置 37℃温育 1 小时。
4. 手工洗板，弃去孔内液体。洗涤液注满各孔，静置 10 秒甩干，重复三次
后拍干；洗板机洗板，选择洗涤三次程序，洗板后拍干。
5. 每孔加显色剂 A 液 50μl，显色剂 B 液 50μl，振荡混匀后，37℃避光显
色 15 分钟，每孔加终止液 50μl。
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6. 用酶标仪读数，取波长 450nm，先用空白孔调零点，然后测定各孔 OD值。
数据处理 数据处理 数据处理 数据处理
1. 手工作图：用双对数坐标纸，以标准品浓度为横轴，以对应的 0D值为纵
轴，画出平滑曲线或直线，在曲线上按照待测血清 OD值找到对应的浓度
值。
2. 计算机：使用线性拟合功能，应将标准品 S1-S5 的浓度取对数 （Log（浓度））
作为 X，将对应的 OD 值减去空白对照孔 OD 值后取对数（Log（OD 值
-NSB））作为 Y，进行线性拟合。再从拟合线上计算出待测血清浓度。
注意事项 注意事项 注意事项 注意事项
1. 从冷藏环境中取出的试剂盒内全部瓶装试剂及所需预包被板条应置室温
（18-25℃）平衡 30 分钟后方可使用，余者应及时封好口，放回 2-8℃中避光
保存，以备后用。
2. 使用前试剂应摇匀。
3. 结果判断须在反应终止后 10 分钟内完成。
4. 不同批号的试剂不可混用。
5. 加样时应注意避免所用各试剂及样品之间的交又污染。
6. 操作时，试剂盒内每种试剂各使用一个吸头，每一种标准品使用一个吸头，
每一个样品各使用一个吸头，吸头一次性使用。
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