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Human P-selectin ELISA Kit
(96 T)
? This immunoassay kit allows for the in vitro quantitative determination of human
P-selectin 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
P-selectin is a cell adhesion molecule (CAM) on the surfaces of activated
endothelial cells, which line the inner surface of blood vessels, and
activated platelets. In unactivated endothelial cells, it is stored in granules
Weibel-Palade bodies, and α-granules in unactivated platelets. P-selectin
plays an essential role in the initial recruitment of leukocytes (white blood
cells) to the site of injury during inflammation.
Thrombin is one trigger which can stimulate endothelial-cell release of
P-selectin and recent studies suggest an additional Ca2+-independent
pathway involved in release of P-selectin. Ligands for P-selectin on
eosinophils and neutrophils are similar sialylated, protease-sensitive,
endo-beta-galactosidase-resistant structures, clearly different than those
reported for E-selectin, and suggest disparate roles for P-selectin and
E-selectin during recruitment during inflammatory responses. P-selectin
attaches to the actin cytoskeleton through anchor proteins that are still
poorly characterized.
PRINCIPLE OF THE ASSAY
The microtiter plate provided in this kit has been pre-coated with an
antibody specific to P-selectin. Standards or samples are then added to the
appropriate microtiter plate wells with a biotin-conjugated polyclonal
antibody preparation specific for P-selectin 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 P-selectin,
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 P-selectin in the samples is then determined by comparing
the O.D. of the samples to the standard curve.
DETECTION RANGE
0.94 ng/ml-60 ng/ml. The standard curve concentrations used for the
ELISA’s were 60 ng/ml, 30 ng/ml,15 ng/ml, 7.5 ng/ml, 3.75 ng/ml, 1.88
ng/ml, 0.94 ng/ml.
SPECIFICITY
This assay recognizes recombinant and natural human P-selectin. No
significant cross-reactivity or interference was observed.
SENSITIVITY
The minimum detectable dose of human P-selectin is typically less than
0.24 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
Standard 2
Sample Diluent 2 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
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. 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.
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.
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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 Centrifuge the standard vial at 6000-10000rpm for 30s.
Reconstitute the Standard with 1.0 ml of Sample Diluent. This
reconstitution produces a stock solution of 60 ng/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 (60
ng/ml). The Sample Diluent serves as the zero standard (0 ng/ml.).
Prepare fresh for each assay. Use within 4 hours and discard after use.
3. Biotin-antibody Centrifuge the vial before opening. Dilute to the
working concentration using Biotin-antibody Diluent(1:100),
respectively.
4. HRP-avidin Centrifuge the vial before opening. Dilute to the working
concentration using HRP-avidin Diluent(1:100), respectively.
OTHER SUPPLIES REQUIRED
? Microplate reader capable of measuring absorbance at 450 nm, with
the correction wavelength set at 540 nm or 570 nm.
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? 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 immediately 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 30 minutes of
collection. Assay immediately 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.
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1. Serum and plasma samples require a 20-fold dilution into Sample
Diluent. The suggested 20-fold dilution can be achieved by adding 15μl
sample to 285μl of Sample Diluent. The recommended dilution factor is
for reference only. The optimal dilution factor should be determined by
users according to their particular experiments.
2. Add 100μl of Standard, Blank, or Sample per well. Cover with the
adhesive strip. Incubate for 2 hours at 37°C.
3. Remove the liquid of each well, don’t wash.
4. 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.
5. Aspirate each well and wash, repeating the process three times for a
total of three washes. Wash: Fill each well with Wash Buffer (200μl) and
let it stand for 2 minutes, then remove the liquid by flicking the plate
over a sink. The remaining drops are removed by patting the plate on a
paper towel. Complete removal of liquid at each step is essential to
good performance.
6. 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.
7. Repeat the aspiration and wash three times as step 4.
8. Add 90μl of TMB Substrate to each well. Incubate for 10-30 minutes at
37°C. Keeping the plate away from drafts and other temperature
fluctuations in the dark.
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9. 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.
10. Determine the optical density of each well within 30 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, 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 x-axis
against the concentration on the y-axis and draw a best fit curve through the
points on the graph. The data may be linearized by plotting the log of the
P-selectin 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.
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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 Standard 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 Standard 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 Immunoassay, the possibility of
interference cannot be excluded.
TECHNICAL HINTS
? Centrifuge vials before opening to collect contents.
? 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.
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? 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 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.
? 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P 选择素(P-Selectin)酶联免疫分析
试剂盒使用说明书
本试剂盒仅供研究使用
检测范围：0.94 ng/ml – 60 ng/ml
最低检测限：0.24 ng/ml
特异性：本试剂盒可同时检测天然或重组的人P-selectin，且与其他相关
蛋白无交叉反应。
有效期：6 个月
预期应用：ELISA 法定量测定人血清、血浆或其它相关生物液体中
P-selectin 含量。
说明
1 试剂盒保存：未开封的试剂盒应储存于2-8℃；开封后的酶标板应与干燥
剂一起储存于铝箔袋中置于2-8℃保存。仅在此出储存条件下，产品在有
效期内可正常使用。
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
用纯化的抗体包被微孔板，制成固相载体，往包被抗P-selectin 抗体的
微孔中依次加入标本或标准品、生物素化的抗P-selectin 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
P-selectin 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
1. 酶联板(Assay plate )： 一块（96 孔）。
2. 标准品（Standard）： 2 瓶(冻干品)。
3. 样品稀释液(Sample Diluent)： 2×20ml/瓶。
4. 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（HRP-avidin Diluent） 1×10ml/瓶。
6. 生物素标记抗体（Biotin-antibody）： 1×120μl/瓶（1：100）。
7. 辣根过氧化物酶标记亲和素（HRP-avidin）： 1×120μl/瓶（1：100）。
8. 底物溶液（TMB Substrate）： 1×10ml/瓶。
9. 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（Stop Solution）： 1×10ml/瓶。
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
1. 血清：全血标本请于室温放置2 小时或4℃过夜后于1000g 离心20 分钟，
取上清即可立即检测；或进行分装，并将标本放于-20℃或-80℃保存，但
应避免反复冻融。解冻后的样品应再次离心，然后检测。
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 g 离心15 分钟，取上清即可立即检测；或进行分装，并将标本放于
-20℃或-80℃保存，但应避免反复冻融。解冻后的样品应再次离心，然后
检测。
注：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。
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标准品的稀释原则：2 瓶，使用前于6000-10000rpm 离心30 秒。 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60 ng/ml，做系列倍比稀释后，分别稀释60 ng/ml,
30 ng/ml,15 ng/ml, 7.5 ng/ml, 3.75 ng/ml, 1.88 ng/ml, 0.94 ng/ml，样品稀释
液直接作为标准浓度0 ng/ml，临用前15 分钟内配制，用完丢弃，下次检测
使用新鲜配置的标准品。
如配制30 ng/ml 标准品：取0.5ml（不要少于0.5ml）60 ng/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：
打开瓶盖前请离心，收集瓶壁上的溶液。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：
打开瓶盖前请离心，收集瓶壁上的溶液。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
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。加样时，枪头应直接对
准液面，切勿沿孔壁加样。
1. 血清，血浆样本用样本稀释液进行1:20 倍稀释后进行检测，具体操作如
下：取15μl 样本加入到285μl 的样本稀释液（1:20 稀释）中混匀，得
到的即为1:20 倍稀释后的样本。此推荐稀释倍数仅供参考，用户应根据
实验自行确定其最优稀释倍数。
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2. 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。
3. 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。
4. 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。
5. 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。
6. 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。
7. 依序每孔加底物溶液90μl，37℃避光显色（20-30 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 孔显色不明显，即可终止）。
8. 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。
9. 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
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
液及终止液。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
请从我们的网站下载专业软件"Curve Exert 1.3"，并根据提示制作标准曲线。
以标准物的浓度为纵坐标（对数坐标），OD 值为横坐标（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
1. 本操作说明也适用于48T 试剂盒， 48T 试剂盒中酶联板、标准品、生物
素标记抗体及辣根过氧化物酶标记亲和素减半。
2. 当混合蛋白溶液时应尽量轻缓，避免起泡。
3. 洗涤过程非常重要，不充分的洗涤易造成假阳性。
4. 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。
5. 请每次测定的同时做标准曲线，最好做复孔。
6. 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。
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. 底物请避光保存。
9. 不要用其它生产厂家的试剂替换试剂盒中的试剂。