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Human C-Reactive
Protein,CRP
ELISA Kit
This immunoassay kit allows for the in vitro quantitative determination of human
CRP 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
C-reactive protein (CRP) is a protein found in the blood in response to
inflammation (an acute-phase protein). CRP is synthesized by the liver in
response to factors released by fat cells (adipocytes). It is a member of the
pentraxin family of proteins. CRP is a member of the class of acute-phase
reactants as its levels rise dramatically during inflammatory processes
occurring in the body. This increment is due to a rise in the plasma
concentration of IL-6, which is produced predominantly by macrophages
as well as adipocytes. CRP binds to phosphocholine on microbes. It is
thought to assist in complement binding to foreign and damaged cells and
enhances phagocytosis by macrophages, which express a receptor for
CRP. It is also believed to play another important role in innate immunity, as
an early defense system against infections. CRP rises up to 50,000-fold in
acute inflammation, such as infection. It rises above normal limits within 6
hours, and peaks at 48 hours. Its half-life is constant, and therefore its
level is mainly determined by the rate of production (and hence the severity
of the precipitating cause). Serum amyloid A is a related acute-phase
marker that responds rapidly in similar circumstances. To measure the CRP
level, a "high-sensitivity" CRP or hs-CRP test needs to be performed and
analyzed by a laboratory. This is an automated blood test designed for
greater accuracy in measuring low levels of CRP, which allows the
physician to assess cardiovascular risk.
PRINCIPLE OF THE ASSAY
The microtiter plate provided in this kit has been pre-coated with an
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antibody specific to CRP. Standards or samples are then added to the
appropriate microtiter plate wells with a HRP-conjugated antibody
preparation specific for CRP 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 CRP, HRP-conjugated antibody 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 CRP in the samples is then determined by comparing the
O.D. of the samples to the standard curve.
DETECTION RANGE
0.625 ng/ml-40 ng/ml. The standard curve concentrations used for the
ELISA’s were 40 ng/ml, 20 ng/ml, 10 ng/ml,5 ng/ml, 2.5 ng/ml, 1.25 ng/ml,
0.625ng/ml.
SPECIFICITY
This assay recognizes recombinant and natural human CRP. No significant
cross-reactivity or interference was observed.
SENSITIVITY
The minimum detectable dose of human CRP is typically less than 0.156
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 4 x 20 ml
HRP-conjugate Diluent 1 x 10 ml
HRP-conjugate 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 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. 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 for 30min.
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.
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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 40 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 (40
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. HRP-conjugate Dilute to the working concentration using
HRP-conjugate Diluent(1:100), respectively. The suggested 100-fold
dilution can be achieved by adding 10 uL HRP-conjugate to 990uL of
HRP-conjugate Diluent for 1ml working solution.
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 immediately 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 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.
If samples generate values higher than the highest standard, dilute the samples
with the Sample Diluent and repeat the assay.
SAMPLE PREPARTION
Normal human serum samples require a 1000-fold dilution into Sample
Diluent. The suggested 1000-fold dilution can be achieved by adding 5µl
sample to 95µl of Sample Diluent. Complete the 1000-fold dilution by
adding 5µl of this solution to 245µl 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.
As CRP levels increase widely, the patient serum samples are
recommended to dilute 1:1000-1:30000 times before test. The
recommended dilution factor is for reference only. The optimal dilution
factor should be determined by users according to their particular
experiments.
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. Add 100µl Sample Diluent serves as the zero standard, Add 100µl of
Standard or Sample per well. Cover with the adhesive strip. Incubate for
60min at 37°C.
2. 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.
3. Add 100µl of HRP-conjugate working solution to each well. Incubate
for 60min at 37°C. HRP-conjugate working solution may appear cloudy.
Warm up to room temperature and mix gently until solution appears
uniform.
4. Repeat the aspiration and wash five times as before.
5. Add 90µl of TMB Substrate to each well. Incubate for 20 minutes at
37°C. Keeping the plate away from drafts and other temperature
fluctuations in the dark.
6. 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.
7. 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.
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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
CRP 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.
Any variation in 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
When mixing or reconstituting protein solutions, always avoid foaming.
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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 人人 人 C 反应蛋白 反应蛋白 反应蛋白 反应蛋白(CRP)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-E07921h
检测范围 检测范围 检测范围 检测范围： ：： ：0.625 ng/ml – 40 ng /ml
最低检测限 最低检测限 最低检测限 最低检测限： ：： ：0.156 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人 CRP，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法定量测定人血清、血浆中 CRP 含量。
说明 说明 说明 说明
1. 试剂盒保存： 2-8℃。
2. 中、英文说明书可能会有不一致之处，请以英文说明书为准。
3. 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
用纯化的抗体包被微孔板，制成固相载体，往包被抗 CRP 抗体的微孔中
加入标本或标准品、HRP 标记的抗 CRP 抗体，经过彻底洗涤后用底物显色。
底物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 CRP 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 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. 酶结合物 酶结合物 酶结合物 酶结合物（ （（ （HRP-conjugate） ）） ） : 1 x 120µl /瓶。
4. 酶结合物稀释液 酶结合物稀释液 酶结合物稀释液 酶结合物稀释液(HRP-conjugate Diluent) : 1×10ml/瓶。
5. 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。
6. 底物溶液 底物溶液 底物溶液 底物溶液 （ （（ （TMB Substrate） ）） ）： 1×10ml/瓶。
7. 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。
8. 终止液 终止液 终止液 终止液（ （（ （Stop Solution） ）） ）： 1×10ml/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，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。
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： ：： ：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。 。。 。
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样本稀释倍数 样本稀释倍数 样本稀释倍数 样本稀释倍数
正常人血清推荐 1:1000 倍稀释后用于检测。具体操作如下：取 5µl 样本加入
到 95µl 的样本稀释液（1:20 稀释）中混匀，再从上述稀释液中取 5µl加入到
245µl 样本稀释液 （1:50 稀释）中混匀。得到的即为 1:1000 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。
因发病时 CRP 含量有不同程度增高，推荐对病人血清进行 1:1000-1:30000
倍稀释。各实验室应当通过预实验确定最合适的稀释倍数。
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，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 40ng/ml，做系列倍比稀释后，分别稀释 40 ng/ml,
20 ng/ml, 10 ng/ml,5 ng/ml, 2.5 ng/ml, 1.25 ng/ml, 0.625 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 20ng/ml 标准品：取 0.5ml（不要少于 0.5ml）40ng/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。
酶结合 酶结合 酶结合 酶结合物 物物 物的稀释原则 的稀释原则 的稀释原则 的稀释原则： ：： ：
打开瓶盖前请离心，收集瓶壁上的溶液。临用前用酶结合物稀释液 酶结合物稀释液 酶结合物稀释液 酶结合物稀释液稀释，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 酶结合物 酶结合物 酶结合物 酶结合物加990µl 酶结合物稀释液 酶结合物稀释液 酶结合物稀释液 酶结合物稀释液 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。
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1. 加样：分别设标准孔、待测样品孔。加 100µl 样品稀释液作为标准品 S0
孔。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 60 分钟。
2. 温育后，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， 最后用力在洗水纸上拍干。
3. 每孔加酶结合物 100µl，空白孔不加。混匀，37℃，60 分钟。
4. 温育后，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，最后用力在洗水纸上拍干。
5. 依序每孔加底物溶液 90µl，37℃避光显色20分钟。
6. 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。
7. 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. 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。
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。
洗板方法 洗板方法 洗板方法 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。
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计算 计算 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 注意事项 注意事项
1. 当混合蛋白溶液时应尽量轻缓，避免起泡。
2. 洗涤过程非常重要，不充分的洗涤易造成假阳性。
3. 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。
4. 请每次测定的同时做标准曲线，最好做复孔。
5. 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。
6. 底物请避光保存。
7. 不要用其它生产厂家的试剂替换试剂盒中的试剂。
