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Rabbit tumor necrosis factor α
(TNF-α) ELISA Kit
Catalog No. CSB-E06998Rb
(96 tests)
This immunoassay kit allows for the in vitro quantitative determination of rabbit
TNF-α 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.
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INTRODUCTION
The prototype ligand of the TNF superfamily, TNF-α/TNFSF1A, is a
pleiotropic cytokine that plays a central role in inflammation and apoptosis.
It is synthesized as a 26 kDa, type II transmembrane protein that is 233
amino acids in length. It contains a 30 amino acid (aa) cytoplasmic domain,
a 26 aa transmembrane segment, and a 177 aa extracellular region.
TNF-αis assembled intracellularly to form a transmembrane,
non-covalently-linked homotrimeric protein. The 157 aa residue soluble
form of TNF-α(sTNF-αis released from the C-terminus of the
transmembrane protein through the activity of TNF-α-converting enzyme
(TACE), a membrane -bound disintegrin metalloproteinase. Human cells
known to express TNF-αinclude B cells, colonic columnar epithelial cells,
NK and CD3+CD56+ hepatic natural T cells, macrophages, monocytes and
monocyte-derived dendritic cells, CD4+ and CD8+ T cells, mast cells,
neutrophils, keratinocytes, plasma cells, and adipocytes.
PRINCIPLE OF THE ASSAY
The microtiter plate provided in this kit has been pre-coated with an
antibody specific to TNF-α. Standards or samples are then added to the
appropriate microtiter plate wells with a biotin-conjugated polyclonal
antibody preparation specific for TNF-α and Avidin conjugated to
Horseradish Peroxidase (HRP) is added to each microplate well and
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incubated. Then a TMB (3,3’5, 5’ tetramethyl-benzidine) substrate solution
is added to each well. Only those wells that contain TNF-α,
biotin-conjugated antibody and enzyme-conjugated Avidin will exhibit a
change in color. 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 TNF-α in the samples is then determined by comparing the
O.D. of the samples to the standard curve.
DETECTION RANGE
78 pg/ml-5000 pg/ml. The standard curve concentrations used for the
ELISA’s were 5000 pg/ml, 2500 pg/ml, 1250 pg/ml, 625 pg/ml, 312 pg/ml,
156 pg/ml, 78 pg/ml.
SPECIFICITY
This assay recognizes recombinant and natural rabbit TNF-α. No significant
cross-reactivity or interference was observed.
SENSITIVITY
The minimum detectable dose of rabbit TNF-α is typically less than 19.5
pg/ml.
The sensitivity of this assay, or Lower Limit of Detection (LLD) was defined
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as the lowest protein concentration that could be differentiated from zero.
MATERIALS PROVIDED
Reagent Quantity
Assay plate 1
Standard 2
Sample Diluent 1 x 20 ml
Biotin-antibody Diluent 1 x 10 ml
HRP-avidin Diluent 1 x 10 ml
Biotin-antibody 1 x 120l
HRP-avidin 1 x 120l
Wash Buffer
1 x 20 ml
(25×concentrate)
TMB Substrate 1 x 10 ml
Stop Solution 1 x 10 ml
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.
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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.
REAGENT PREPARATION
Bring all reagents to room temperature before use.
1. Wash Buffer If crystals have formed in the concentrate, warm to
room temperature and mix gently until the crystals have completely
dissolved. Dilute 20 ml of Wash Buffer Concentrate into deionized or
distilled water to prepare 500 ml of Wash Buffer.
2. Standard Reconstitute the Standard with 1.0 ml of Sample Diluent.
This reconstitution produces a stock solution of 5000 pg/ml. Allow the
standard to sit for a minimum of 15 minutes with gentle agitation prior to
making serial dilutions. The undiluted standard serves as the high
standard (5000 pg/ml). The Sample Diluent serves as the zero
standard (0 pg/ml).
3. Biotin-antibody Dilute to the working concentration specified on the
vial label using Biotin-antibody Diluent(1:100), respectively.
4. HRP-avidin Dilute to the working concentration specified on the vial
label using HRP-avidin Diluent(1:100), respectively.
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
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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
Cell Culture Supernates Remove particulates by centrifugation and
assay immediately or aliquot and store samples at-20° C. Avoid
repeated freeze-thaw cycles.
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 immediately 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 immediately 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. Add 100l of Standard, Blank, or Sample per well. Cover with the
adhesive strip. Incubate for 2 hours at 37° C.
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2. Remove the liquid of each well, don’t wash.
3. Add 100l of Biotin-antibody working solution to each well. Incubate
for 1 hour at 37°C. Biotin-antibody working solution may appear
cloudy. Warm to room temperature and mix gently until solution
appears uniform.
4. Aspirate each well and wash, repeating the process three times for a
total of three washes. Wash by filling each well with Wash Buffer (350l)
using a squirt bottle, multi-channel pipette, manifold dispenser or
autowasher. 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.
5. Add 100l of HRP-avidin working solution to each well. Cover the
microtiter plate with a new adhesive strip. Incubate for 1 hours at 37° C.
6. Repeat the aspiration and wash three times as step 4.
7. Add 90l of TMB Substrate to each well. Incubate for 30 minutes at
37°C. Keeping the plate away from drafts and other temperature
fluctuations in the dark.
8. Add 50l of Stop Solution to each well. If color change does not
appear uniform, gently tap the plate to ensure thorough mixing.
9. Determine the optical density of each well within 30 minutes, using a
microplate reader set to 450 nm.
CALCULATION OF RESULTS
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Average the duplicate readings for each standard, control, and sample and
subtract the average zero standard optical density. 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
TNF-α 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.
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.
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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.
TECHNICAL HINTS
When mixing or reconstituting protein solutions, always avoid foaming.
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.
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.
To ensure accurate results, proper adhesion of plate sealers during
incubation steps is necessary.
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.
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.
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兔子肿瘤坏死因子 兔子肿瘤坏死因子 兔子肿瘤坏死因子 兔子肿瘤坏死因子 α(TNF-α)酶联免疫分析 酶联免疫分析 酶联免疫分析 酶联免疫分析
试剂盒使用说明书 试剂盒使用说明书 试剂盒使用说明书 试剂盒使用说明书
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-E06998Rb
检测范围 检测范围 检测范围 检测范围： ：： ：78 pg/ml - 5000 pg/ml
最低检测限 最低检测限 最低检测限 最低检测限： ：： ：19.5 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兔子 TNF-α，且与其他相关蛋
白无交叉反应。
有效期 有效期 有效期 有效期： ：： ：6 个月
预期应用 预期应用 预期应用 预期应用： ：： ：ELISA 法定量测定兔子血清、血浆或其它相关生物液体中
TNF-α 含量。
说明 说明 说明 说明
1. 试剂盒保存：-20℃（较长时间不用时）；2-8℃（频繁使用时）。
2. 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。
3. 中、英文说明书可能会有不一致之处，请以英文说明书为准。
4. 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
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用纯化的抗体包被微孔板，制成固相载体，往包被抗 TNF-α 抗体的微孔
中依次加入标本或标准品、生物素化的抗 TNF-α 抗体、HRP 标记的亲和素，
经过彻底洗涤后用底物 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最终的黄色。颜色的深浅和样品中的TNF-α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。
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. 酶联板 酶联板 酶联板 酶联板(Assay plate )： 一块 （96孔）。
2. 标准品 标准品 标准品 标准品 （Standard）： 2 瓶(冻干品)。
3. 样品稀释液 样品稀释液 样品稀释液 样品稀释液(Sample Diluent)： 1×20ml/瓶。
4. 生物素标记抗体稀释液 生物素标记抗体稀释液 生物素标记抗体稀释液 生物素标记抗体稀释液（ （（ （Biotin-antibody Diluent） ）） ）： 1×10ml/瓶。
5. 辣根过氧化物酶标记亲和素稀释液 辣根过氧化物酶标记亲和素稀释液 辣根过氧化物酶标记亲和素稀释液 辣根过氧化物酶标记亲和素稀释液（ （（ （HRP-avidin Diluent） ）） ）：1×10ml/瓶。
6. 生物素标记抗体 生物素标记抗体 生物素标记抗体 生物素标记抗体 （ （（ （Biotin-antibody） ）） ）： 1×120l/瓶（1： 100）。
7. 辣根过氧化物酶标记亲和素 辣根过氧化物酶标记亲和素 辣根过氧化物酶标记亲和素 辣根过氧化物酶标记亲和素 （ （（ （HRP-avidin） ）） ）： 1×120l/瓶（1： 100）。
8. 底物溶液 底物溶液 底物溶液 底物溶液 （ （（ （TMB Substrate） ）） ）： 1×10ml/瓶。
9. 浓洗涤液 浓洗涤液 浓洗涤液 浓洗涤液（ （（ （Wash Buffer） ）） ） 1×20ml/瓶，使用时每瓶用蒸馏水稀释 25 倍。
10. 终止液 终止液 终止液 终止液 （ （（ （Stop Solution） ）） ）： 1×10ml/瓶（2N H2SO4）。
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，最好用多通道移液器
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。
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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。
3. 细胞培养物上清或其它生物标本：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。
注 注注 注： ：： ：标本溶血会影响最后检测结果 标本溶血会影响最后检测结果 标本溶血会影响最后检测结果 标本溶血会影响最后检测结果， ，， ，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。 。。 。
标本的稀释原则 标本的稀释原则 标本的稀释原则 标本的稀释原则： ：： ：
首先通过文献检索的方式了解待测样本的大致含量，确定适当的稀释倍数。
只有稀释至标准曲线的范围内，检测的结果才是准确的。稀释的过程中，应
做好详细的记录。最后计算浓度时，稀释了“N”倍，标本的浓度应再乘以“N”。
标准品的稀释原则 标准品的稀释原则 标准品的稀释原则 标准品的稀释原则： ：： ：2 瓶，每瓶临用前以样品稀释液稀释至 1ml，盖好
后静置 10 分钟以上，然后反复颠倒/搓动以助溶解，其浓度为 5000 pg/ml，
做系列倍比稀释后，分别稀释 5000 pg/ml，2500 pg/ml，1250 pg/ml，625
pg/ml，312 pg/ml，156 pg/ml，78 pg/ml，样品稀释液直接作为标准浓度 0
pg/ml，临用前 15 分钟内配制。
如配制 2500 pg/ml标准品：取 0.5ml（不要少于 0.5ml）5000 pg/ml的上述
标准品加入含 0.5ml 样品稀释液的 Eppendorf 管中，混匀即可，其余浓度以
此类推。
生物素标记抗体的稀释原则 生物素标记抗体的稀释原则 生物素标记抗体的稀释原则 生物素标记抗体的稀释原则： ：： ：
临用前以生物素标记抗体稀释液稀释，稀释前根据预先计算好的每次实验所
需的总量配制（每孔 100l），实际配制时应多配制 0.1-0.2ml。如 10l 生物
素标记抗体加 990l 生物素标记抗体稀释液的比例配制，轻轻混匀，在使用
前一小时内配制。
辣根过氧化物酶标记亲和素的稀释原则 辣根过氧化物酶标记亲和素的稀释原则 辣根过氧化物酶标记亲和素的稀释原则 辣根过氧化物酶标记亲和素的稀释原则： ：： ：
临用前以辣根过氧化物酶标记亲和素稀释液稀释，稀释前根据预先计算好的
每次实验所需的总量配制（每孔 100l），实际配制时应多配制 0.1-0.2ml。如
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10l 辣根过氧化物酶标记亲和素加 990l 辣根过氧化物酶标记亲和素稀释
液 的比例配制，轻轻混匀，在使用前一小时内配制。
操作步骤 操作步骤 操作步骤 操作步骤
实验开始前，请提前配置好所有试剂，试剂或样品稀释时，均需混匀，混匀
时尽量避免起泡。每次检测都应该做标准曲线。如样品浓度过高时，用样品
稀释液进行稀释，以使样品符合试剂盒的检测范围。
1. 加样：分别设空白孔、标准孔、待测样品孔。空白孔加样品稀释液 100l，
余孔分别加标准品或待测样品 100l，注意不要有气泡，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，酶标板加上盖或覆膜，
37℃反应 120 分钟。
为保证实验结果有效性，每次实验请使用新的标准品溶液。
2. 弃去液体，甩干，不用洗涤。每孔加生物素标记抗体工作液 100l （取 1l
生物素标记抗体加 99l 生物素标记抗体稀释液的比例配制，轻轻混匀，
在使用前一小时内配制），37 ,60 ℃ 分钟。
3. 温育 60 分钟后，弃去孔内液体，甩干，洗板 3 次，每次浸泡 1-2分钟，
350l/每孔，甩干。
4. 每孔加辣根过氧化物酶标记亲和素工作液（同生物素标记抗体工作液）
100l，37℃，60 分钟。
5. 温育 60 分钟后，弃去孔内液体，甩干，洗板 5 次，每次浸泡 1-2分钟，
350l/每孔，甩干。
6. 依序每孔加底物溶液 90l，37℃避光显色（ （（ （30 分钟内，此时肉眼可见标
准品的前 3-4 孔有明显的梯度蓝色，后 3-4 孔梯度不明显，即可终止）。
7. 依序每孔加终止溶液 50l，终止反应（此时蓝色立转黄色）。终止液的加
入顺序应尽量与底物液的加入顺序相同。为了保证实验结果的准确性，底
物反应时间到后应尽快加入终止液。
8. 用酶联仪在 450nm 波长依序测量各孔的光密度（OD 值）。 在加终止液
后 15 分钟以内进行检测。
实验备注 实验备注 实验备注 实验备注
1. 本操作说明适用于 本操作说明适用于 本操作说明适用于 本操作说明适用于 48T 试剂盒 试剂盒 试剂盒 试剂盒， ，， ，但 但但 但 48T 试剂盒所有试剂减半 试剂盒所有试剂减半 试剂盒所有试剂减半 试剂盒所有试剂减半。 。。 。
2. 用户在初次使用试剂盒时，应将各种试剂管离心数分钟，以便试剂集中到
管底。
3. 每次实验留一孔作为空白调零孔，该孔不加任何试剂，只是最后加底物溶
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液及 2N H2SO4。测量时先用此孔调 OD值至零。
4. 为防止样品蒸发，试验时将反应板放于铺有湿布的密闭盒内，酶标板加上
盖或覆膜。
5. 未使用完的酶标板或者试剂，请于 2-8℃保存。标准品、生物素标记抗体
工作液、辣根过氧化物酶标记亲和素工作液请依据所需的量配置使用。请
勿重复使用已稀释过的标准品、生物素标记抗体工作液或、辣根过氧化物
酶标记亲和素工作液。
6. 建议检测样品时均设双孔测定，以保证检测结果的准确性。
