(sTRAIL)人可溶性肿瘤坏死因子相关凋亡诱导配体(sTRAIL)Elisa试剂盒说明书

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Human Soluble Tumor necrosis

Factor-related Apoptosis

Inducing Ligand （（sTRAIL））

（（））

ELISA KIT

Catalog No. CSB-E04750h

(96T)

This immunoassay kit allows for the in vitro quantitative determination of human

sTRAIL concentrations in cell culture supernates, 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/

E-mail: cusabio@cusabio.com cusabio@cusabio.cn

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INTRODUCTION

TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) is a

recently identified member of the TNF gene superfamily. Five different

receptors have been identified for TRAIL. Two receptors, DR4 and DR5, are

transmembrane proteins containing death domain similar to FAS and other

TNF family receptors. Two other receptors, DcR1 and DcR2, act like decoy

proteins for TRAIL binding because they lack the death domain. TRAIL can

also bind, though weakly, to osteoprotegrin (OPG), a soluble receptor,

which plays a role in osteoclastogenesis. TRAIL induces apoptosis in

various tumor cell lines, whereas most primary cells seem to be resistant.

TRAIL-mediated apoptosis occurs following its binding to DR4 or DR5

receptors. The mechanism of apoptosis involves activation of caspase-8

and subsequent activation of effector caspases. Also, NF- κB and JNK

activation play a role in the TRAIL signaling pathway.TRAIL expression is

detectable in many normal organs and tissues. Several studies suggest that

TRAIL may play a physiological role by contributing to immune privilege,

normal cellular development, and inhibition of autoimmune responses. The

expression of TRAIL is upregulated in activated T cells, B cells, NK cells,

monocytes and macrophages. LPS activation stimulates the release of

soluble TRAIL from monocytes and macrophages, suggesting a role of

TRAIL in their cytotoxic/phagocytic function. Also, TRAIL expression is

increased in transformed cell lines and various diseases including cancer

and autoimmune disorders. TRAIL may also be involved in

activation-induced T cell death during HIV infection. Certain anti-cancer

agents also upregulate TRAIL and TRAIL receptor expression on tumor

cells, thus sensitizing cells to apoptosis.

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PRINCIPLE OF THE ASSAY

The microtiter plate provided in this kit has been pre-coated with an

antibody specific to sTRAIL. Standards or samples are then added to the

appropriate microtiter plate wells with a biotin-conjugated polyclonal

antibody preparation specific for sTRAIL. and Avidin conjugated to

Horseradish Peroxidase (HRP) is added to each microplate well and

incubated. Then a TMB (3,3’5, 5’ tetramethyl-benzidine) substrate solution

is added to each well. Only those wells that contain sTRAIL,

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 sTRAIL. in the samples is then determined by comparing

the O.D. of the samples to the standard curve.

DETECTION RANGE

15.6 pg/ml-1000 pg/ml. The standard curve concentrations used for the

ELISA’s were 1000 pg/ml, 500 pg/ml, 250 pg/ml,125 pg/ml, 62.5 pg/ml, 31.2

pg/ml, 15.6 pg/ml.

SPECIFICITY

This assay recognizes recombinant and natural human sTRAIL No

significant cross-reactivity or interference was observed.

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SENSITIVITY

The minimum detectable dose of human sTRAIL is typically less than 3.9

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

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 120μl

HRP-avidin 1 x 120μl

1 x 20 ml

Wash Buffer

(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. The test kit may be used

throughout the expiration date of the kit. Refer to the package label for

the expiration date.

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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.

REAGENT PREPARATION

Bring all reagents to room temperature before use.

1. Wash Buffer If crystals have formed in the concentrate, warm up 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 1000 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 (1000 pg/ml). The Sample Diluent serves as the zero

standard (0 pg/ml).

3. Biotin-antibody Dilute to the working concentration using

Biotin-antibody Diluent(1:100), respectively.

4. HRP-avidin Dilute to the working concentration 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.

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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

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 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.

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1. Add 100μl of Standard, Blank, or Sample per well. Cover with the

adhesive strip. Incubate for 2 hours at 37° C.

2. Remove the liquid of each well, don’t wash.

3. Add 100μl of Biotin-antibody working solution to each well. Incubate

for 1 hour at 37°C. Biotin-antibody working solution may appear

cloudy. Warm up 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 (200μl)

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 100μl of HRP-avidin working solution to each well. Cover the

microtiter plate with a new adhesive strip. Incubate for 1 hour at 37° C.

6. Repeat the aspiration and wash three times as step 4.

7. Add 90μl 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 50μl 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.

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CALCULATION OF RESULTS

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

sTRAIL. 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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人可溶性肿瘤坏死因子相关凋亡诱导配体人可溶性肿瘤坏死因子相关凋亡诱导配体(sTRAIL)

人可溶性肿瘤坏死因子相关凋亡诱导配体人可溶性肿瘤坏死因子相关凋亡诱导配体

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

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：：CSB-E04750h

产品编号产品编号：：

检测范围检测范围：：15.6 pg/ml - 1000 pg/ml

检测范围检测范围：：

最低检测限最低检测限：：3.9 pg/ml

最低检测限最低检测限：：

特异性特异性：：本试剂盒可同时检测天然或重组的人sTRAIL，且与其他相关蛋

特异性特异性：：

白无交叉反应。

有效期有效期：：6 个月

有效期有效期：：

预期应用预期应用：：ELISA 法定量测定人血清、血浆、细胞培养上清或其它相关生

预期应用预期应用：：

物液体中sTRAIL 含量。

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

用纯化的抗体包被微孔板，制成固相载体，往包被抗sTRAIL 抗体的微

孔中依次加入标本或标准品、生物素化的抗sTRAIL 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sTRAIL.

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。

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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(Assay plate )：一块（96 孔）。

酶联板酶联板

2. 标准品标准品（Standard）： 2 瓶(冻干品)。

标准品标准品

3. 样品稀释液样品稀释液(Sample Diluent)： 1×20ml/瓶。

样品稀释液样品稀释液

4. 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。

生物素标记抗体稀释液生物素标记抗体稀释液（（））

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（（HRP-avidin Diluent）：） 1×10ml/瓶。

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（（））

6. 生物素标记抗体生物素标记抗体（（Biotin-antibody）：） 1×120μl/瓶（1：100）。

生物素标记抗体生物素标记抗体（（））

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（（HRP-avidin）：） 1×120μl/瓶（1：100）。

辣根过氧化物酶标记亲和素辣根过氧化物酶标记亲和素（（））

8. 底物溶液底物溶液（（TMB Substrate）：） 1×10ml/瓶。

底物溶液底物溶液（（））

9. 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。

浓洗涤液浓洗涤液（（））

10. 终止液终止液（（Stop Solution）：） 1×10ml/瓶（2N H SO4）。

终止液终止液（（）） 2

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

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。

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：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。。

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，，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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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：：2 瓶，每瓶临用前以样品稀释液稀释至1ml，盖好

标准品的稀释原则标准品的稀释原则：：

后静置10 分钟以上，然后反复颠倒/搓动以助溶解，其浓度为1000 pg/ml，

做系列倍比稀释后，分别稀释1000 pg/ml, 500 pg/ml, 250 pg/ml,125 pg/ml,

62.5 pg/ml, 31.2 pg/ml, 15.6 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500pg/ml 标准品：取0.5ml （不要少于0.5ml）1000 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：：

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100μl），实际配制时应多配制0.1-0.2ml。如10μl 生物

素标记抗体加990μl 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：：

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100μl），实际配制时应多配制0.1-0.2ml。如

10μl 辣根过氧化物酶标记亲和素加990μl 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

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。

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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100μl，

余孔分别加标准品或待测样品100μl，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 100μl （取1μl

生物素标记抗体加99μl 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，

200μl/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）

100μl，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，

200μl/每孔，甩干。

6. 依序每孔加底物溶液90μl，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50μl，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

1. 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。

2. 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

液及2N H SO4。测量时先用此孔调OD 值至零。

3. 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。

4. 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。

5. 建议检测样品时均设双孔测定，以保证检测结果的准确性。

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洗板方洗板方法法

手工洗板方法：吸去（不可触及板壁）或甩掉酶标板内的液体；在实验台上

铺垫几层吸水纸，酶标板朝下用力拍几次；将推荐的洗涤缓冲液至少0.3ml

注入孔内，浸泡1-2 分钟。根据需要，重复此过程数次。

自动洗板：如果有自动洗板机，应在熟练使用后再用到正式实验过程中。

计算计算

以标准物的浓度为横坐标（对数坐标），OD 值为纵坐标（普通坐标），在半对

数坐标纸上绘出标准曲线，根据样品的OD 值由标准曲线查出相应的浓度；

再乘以稀释倍数；或用标准物的浓度与OD 值计算出标准曲线的直线回归方

程式，将样品的OD 值代入方程式，计算出样品浓度，再乘以稀释倍数，即

为样品的实际浓度。

注意事项注意事项

1. 底物请避光保存。

2. 当混合蛋白溶液时应尽量轻缓，避免起泡。

3. 请每次测定的同时做标准曲线，最好做复孔。

4. 洗涤过程非常重要，不充分的洗涤易造成假阳性。

5. 不要用其它生产厂家的试剂替换试剂盒中的试剂。

6. 一次加样时间最好控制在5 分钟内，如标本数量多，推荐使用排枪加样。

7. 在配制标准品、检测溶液工作液时，请以相应的稀释液配制，不能混淆。

8. 如标本中待测物质含量过高，请先稀释后再测定，计算时请最后乘以稀释

倍数。

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Notes：：

：：

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