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Rat Estradiol ELISA Kit
Catalog No. CSB-E05110r
（96T）
This immunoassay kit allows for the in vitro quantitative determination of rat Estradiol
concentrations in serum, plasma and other biological fluids.
Expiration date six months from the date of manufacture
FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES.
CUSABIO BIOTECH CO., LTD.
http://www.cusabio.com/ http://www.cusabio.cn/
: cusabio@cusabio.com cusabio@cusabio.cn
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INTRODUCTION
Estradiol （E2 or 17β-estradiol） （also oestradiol） is a sex hormone. During
the reproductive years, most estradiol in women is produced by the
granulosa cells of the ovaries by the aromatization of androstenedione
（produced in the theca folliculi cells） to estrone, followed by conversion
of estrone to estradiol by 17β-hydroxysteroid reductase. Smaller amounts
of estradiol are also produced by the adrenal cortex, and （in men）, by the
testes. Estradiol is not only produced in the gonads: in both sexes,
precursor hormones, specifically testosterone, are converted by
aromatization to estradiol. In particular, fat cells are active to convert
precursors to estradiol, and will continue to do so even after menopause.
Estradiol is also produced in the brain and in arterial walls. Estradiol
enters cells freely and interacts with a cytoplasmic target cell receptor.
When the estrogen receptor has bound its ligand it can enter the nucleus
of the target cell, and regulate gene transcription which leads to
formation of messenger RNA. The mRNA interacts with ribosomes to
produce specific proteins that express the effect of estradiol upon the
target cell. Estradiol binds well to both estrogen receptors, ERα and ERβ,
in contrast to certain other estrogens, notably medications that
preferentially act on one of these receptors. These medications are called
selective estrogen receptor modulators, or SERMs.
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PRINCIPLE OF THE ASSAY
The microtiter plate provided in this kit has been pre-coated with an
goat-anti-rabbit antibody. Standards or samples are then added to the
appropriate microtiter plate wells with a HRP-conjugated Estradiol and
antibody preparation specific for Estradiol and incubated. Then a TMB
（3,3',5,5' tetramethyl-benzidine） substrate solution is added to each well.
The enzyme-substrate reaction is terminated by the addition of a
sulphuric acid solution and the color change is measured
spectrophotometrically at a wavelength of 450 nm ± 2 nm. The
concentration of Estradiol in the samples is then determined by
comparing the O.D. of the samples to the standard curve.
DETECTION RANGE
20 pg/ml-1000 pg/ml. The standard curve concentrations used for the
ELISA’s were 1000 pg/ml, 500 pg/ml, 200 pg/ml, 60 pg/ml, 20 pg/ml.
SPECIFICITY
This assay recognizes recombinant and natural rat Estradiol. No
significant cross-reactivity or interference was observed.
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SENSITIVITY
The minimum detectable dose of rat Estradiol is typically less than 5
pg/ml.
The sensitivity of this assay, or Lower Limit of Detection （LLD） was
defined as the lowest protein concentration that could be differentiated
from zero.
MATERIALS PROVIDED
Reagent Quantity
Assay plate 1（96T）
Standards （S1-S5） 5
HRP-conjugate 1 x 6ml
Antibody 1 x 6 ml
Wash Buffer
1 x 15 ml
（20×concentrate）
Substrate A 1 x 7ml
Substrate B 1 x 7 ml
Stop Solution 1 x 7 ml
Standard Standard 1 Standard 2 Standard 3 Standard 4 Standard 5
Concentration
（pg/ml）
20pg/ml 60pg/ml 200pg/ml 500pg/ml 1000pg/ml
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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 with desiccants to
minimize exposure to damp air. The test kit may be used throughout
the expiration date of the kit. Refer to the package label for the
expiration date.
2. Opened test 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. 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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3. 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.
4. 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.
5. To ensure accurate results, proper adhesion of plate sealers during
incubation steps is necessary. Sealers can not be reused.
6. Substrate Solution should remain colorless until added to the plate.
Keep Substrate Solution protected from light. Substrate Solution
should change from colorless 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.
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 x g.
Remove serum and assay immediay or aliquot and store samples at
-20° C. Avoid repeated freeze-thaw cycles.
Plasma Collect plasma using citrate, EDTA, or heparin as an
anticoagulant. Centrifuge for 15 minutes at 1000 x g within 30
minutes of collection. Assay immediay or aliquot and store
samples at -20° C. 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.
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1. Set a Blank well without any solution. Add 50l of Standard or
Sample per well. Standard need test in duplicate.
2. Add 50l of HRP-conjugate to each well （not to Blank well）, then
50l antibody to each well. Mix well and then incubate for 2 hours at
37°C.
3. Fill each well with Wash Buffer （about 200l）, 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.
4. Add 50l 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 50l of Stop Solution to each well. 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.
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CALCULATION OF RESULTS
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
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
Estradiol 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.
It is important that the Calibrator Diluent selected for the standard
curve be consistent with the samples being assayed.
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If samples generate values higher than the highest standard, dilute the
samples with the appropriate Calibrator Diluent and repeat the assay.
Any variation in Standard Diluent, operator, pipetting technique,
washing technique, incubation time or temperature, and kit age can
cause variation in binding.
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 大鼠 大鼠 大鼠雌二醇 雌二醇 雌二醇 雌二醇酶联免疫 酶联免疫 酶联免疫 酶联免疫试剂盒 试剂盒 试剂盒 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-E05110r
检测范围 检测范围 检测范围 检测范围： ：： ：20 pg/ml -1000 pg/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雌二醇，且与其他相关物质基本
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，血浆及其它相关生物液体中雌二醇
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
采用酶联免疫竞争法检测雌二醇含量。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、辣根过氧化物酶标记的雌二醇以及抗雌二醇抗
体，使之形成包被二抗-抗雌二醇抗体-雌二醇（HRP）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
标本中雌二醇含量。
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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 瓶。
Standard 1 Standard 2 Standard 3 Standard 4 Standard 5
20pg/ml 60pg/ml 200pg/ml 500pg/ml 1000pg/ml
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
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℃ ℃℃ ℃不应超过 不应超过 不应超过 不应超过 1 个月 个月 个月 个月， ，， ，
-80℃ ℃℃ ℃不应超过 不应超过 不应超过 不应超过 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， ，， ，因此溶血标本不宜进行此项检测 因此溶血标本不宜进行此项检测 因此溶血标本不宜进行此项检测 因此溶血标本不宜进行此项检测。 。。 。
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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），再按同样顺序每孔加入抗体
50ul，充分混匀，贴上不干胶封片，置 37℃温育 2 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。
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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