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Rat Testoterone（ （ （ （T） ） ） ）ELISA Kit
Catalog No. CSB-E05100r
（96T）
This immunoassay kit allows for the in vitro quantitative determination of rat
testoterone 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
Testosterone is a steroid hormone from the androgen group. In mammals,
testosterone is primarily secreted in the testes of males and the ovaries of females,
although small amounts are also secreted by the adrenal glands. It is the principal
male sex hormone and an anabolic steroid.
In both men and women, testosterone plays a key role in health and well-being as
well as in sexual functioning. Examples include enhanced libido, increased energy,
increased production of red blood cells and protection against osteoporosis. On
average, an adult human male body produces about forty to sixty times more
testosterone than an adult female body, but females are, from a behavioral
perspective （rather than from an anatomical or biological perspective）, more
sensitive to the hormone. However the overall ranges for male and female are very
wide, such that the ranges actually overlap at the low end and high end
respectively.
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
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microtiter plate wells with a HRP-conjugated testoterone and antibody preparation
specific for testoterone and incubated. Then substrate solutions are 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 testoterone in the samples is
then determined by comparing the O.D. of the samples to the standard curve.
DETECTION RANGE
0.1 ng/ml-25.6 ng/ml. The standard curve concentrations used for the ELISA’s
were 25.6 ng/ml, 6.4 ng/ml, 1.6 ng/ml, 0.39 ng/ml, 0.1 ng/ml.
SPECIFICITY
This assay recognizes recombinant and natural rat testoterone. No significant
cross-reactivity or interference was observed.
SENSITIVITY
The minimum detectable dose of rat testoterone is typically less than 0.06 ng/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.
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MATERIALS PROVIDED
Reagent Quantity
Assay plate 1
Standards （S1-S5） 5 x 0.5 ml
HRP-conjugate 1 x 6 ml
Antibody 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 x7 ml
Standard Standard 1 Standard 2 Standard 3 Standard 4 Standard 5
Concentration
（ng/ml）
0.1 0.39 1.6 6.4 25.6
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. 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.
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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.
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.
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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.
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.
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Plasma Collect plasma using citrate, EDTA, or heparin as an anticoagulant.
Centrifuge for 15 minutes at 1000 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.
1. Set a Blank well without any solution. Add 50µl of Standard or Sample per
well. Standards need test in duplicate.
2. Add 50µl of HRP-conjugate to each well （not to Blank well）, then add 50µl
Antibody to each well. Mix well and then incubate for 1 hour at 37°C.
3. Fill each well with Wash Buffer （about 200µl）, 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 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.
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5. Add 50µl 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.
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 testoterone 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.
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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.
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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大鼠睾酮 大鼠睾酮 大鼠睾酮 大鼠睾酮（T） （T） （T） （T）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-E05100r
检测范围 检测范围 检测范围 检测范围： ：： ：0.1 ng /ml -25.6 ng /ml
zui低检测限 zui低检测限 zui低检测限 zui低检测限： ：： ：0.06 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睾酮，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，血浆及其它相关生物液体中睾酮
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睾酮含量。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睾酮以及抗睾酮抗体，
使之形成包被二抗-抗睾酮抗体- 睾酮（HRP）复合物，标记睾酮的结合量与
标本中的睾酮量成反比。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，反算出待测标本中睾酮含量。
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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。
Standard 1 Standard 2 Standard 3 Standard 4 Standard 5
0.1 ng/ml 0.39 ng/ml 1.6 ng/ml 6.4 ng/ml 25.6 ng/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 需要而未提供的试 需要而未提供的试 需要而未提供的试剂和器材 剂和器材 剂和器材 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 以上标 以上标 以上标本置 本置 本置 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 个个 个
月 月月 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 因此溶血标本不宜进行 因此溶血标本不宜进行 因此溶血标本不宜进行检 检检 检
测 测测 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），再按同样的顺序加入抗体
50ul，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。
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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