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人 C 反應(yīng)蛋白（超敏）定量分析酶聯(lián)免疫檢測試劑盒

本試劑盒僅供科研使用。用于體外定量檢測人血清、血漿或細胞培養(yǎng)上清液中的 CRP 濃度。使用前請仔細閱讀說明書并檢查試劑組分

是否完整。如有產(chǎn)品包裝破損或質(zhì)量投拆，請在收到貨一個月之內(nèi)聯(lián)系我們。

CRP 簡介：

C 反應(yīng)蛋白（CRP）是人體內(nèi)的急性反應(yīng)蛋白，在人體免疫防御反應(yīng)中重要的介導(dǎo)物，屬于穿透素家族的蛋白。CRP 蛋白的前體蛋白是

224 殘基的蛋白，成熟的 CRP 有 206 個搭氨基酸殘基，通過非共價結(jié)合的方式形成五聚體的環(huán)狀結(jié)構(gòu)。

無論是感染還是非感染性的炎癥、組織壞死和惡性腫瘤時，人體血液中的 CRP 蛋白濃度都會急劇升高。在感染，炎癥反應(yīng)及組織壞死

中，人血清中的 CRP 蛋白在 24~48 小時內(nèi)可急劇升高到正常水平的 1000 倍。

在許多種細菌和真菌表面的膽堿磷酸是 CRP 蛋白的配體，CRP 與配體的結(jié)合是依賴 Ca2+的。CRP 也可與受傷細胞的膜、壞死的胞核和

調(diào)亡的細胞等結(jié)合。一旦與受體結(jié)合，CRP 蛋白就會啟動 C1q，從而引起一系列的級聯(lián)反應(yīng)。CRP 蛋白也會與吞噬細胞的表面 Fcγ RI 和 Fcγ

RII 結(jié)合，從而啟動吞噬細胞的吞噬反應(yīng)。

CRP 主要在肝內(nèi)產(chǎn)生，IL-6 是肝實質(zhì)細胞產(chǎn)生 CRP 蛋白的主要介導(dǎo)物，此外 IL-1 和糖皮質(zhì)激素也是 CRP 蛋白產(chǎn)生重要的誘導(dǎo)物。

檢測原理:

本試劑盒采用雙抗體夾心ELISA法檢測樣本中CRP的濃度。CRP捕獲抗體已預(yù)包被于酶標(biāo)板上，當(dāng)加入標(biāo)本或參考品時，其中的CRP會與

捕獲抗體結(jié)合，其它游離的成分通過洗滌的過程被除去。當(dāng)加入與生物素化的抗人CRP抗體后，抗人CRP抗體與CRP接合，形成夾心的免疫復(fù)

合物，其它游離的成分通過洗滌的過程被除去。再加入親和素連接的HRP結(jié)合物，生物素與親和素結(jié)合，其它游離成分通過洗滌的過程被除

去，最后加入顯色劑，若樣本中存在CRP將會形成免疫復(fù)合物，辣根過氧化物酶會催化無色的顯色劑氧化成藍色物質(zhì)，在加入終止液后呈黃

色。通過酶標(biāo)儀檢測，讀其450nm處的OD值，CRP濃度與OD450值之間呈正比，通過參考品繪制標(biāo)準(zhǔn)曲線，對照未知樣本中OD值，即可算出標(biāo)

本中CRP濃度。

人高敏CRP定量分析酶聯(lián)免疫檢測試劑盒組成:

組分 規(guī)格（96T/48T）

人高敏CRP預(yù)包被板 12條/6條

標(biāo)準(zhǔn)品稀釋液 10ml/5ml

人高敏CRP標(biāo)準(zhǔn)品 4支/2支(凍干)

生物素化的抗CRP抗體 10ml/5ml

親和素連接的HRP結(jié)合物 10ml/5ml

濃縮洗滌液 20× 30ml/15ml

TMB底物 10ml/5ml

中止液 5ml/3ml

封板膠紙 3/2張

說明書 1份

標(biāo)本收集:

1.標(biāo)本的收集請按下列流程進行操作；

A.細胞上清標(biāo)本離心去除懸浮物后即可；

B.血清標(biāo)本應(yīng)是自然凝固后，取上清，避免在冰箱中凝固血液；

C.血漿標(biāo)本，推薦用EDTA的方法收集若待測樣本不能及時檢測，

D.標(biāo)本收集后請分裝，凍存于－20℃，避免反復(fù)凍融。2.血清標(biāo)本不應(yīng)添加任何防腐劑或抗凝劑；

3.標(biāo)本應(yīng)清澈透明，檢測前樣本中如有懸浮物應(yīng)通過離心去除。

4.請勿使用溶血，高血脂或污染的標(biāo)本檢測，否則結(jié)果將不準(zhǔn)確。

注：人血清或血漿樣本請用標(biāo)準(zhǔn)品稀釋液做稀釋后再檢測，稀釋比例為200-4500倍。

注意事項:

1.試劑盒請保存在2～8℃。

2.濃縮洗滌液因在低溫下可能有結(jié)晶，請水浴加熱使結(jié)晶完全溶解后再配制工作液。

3.標(biāo)準(zhǔn)品復(fù)溶加樣后，剩余部分請丟棄。

4.底物請勿接觸氧化劑和金屬。

5.加樣時，請及時更換槍頭，避免交叉污染。

6.嚴(yán)禁混用不同批號的試劑盒組份。

7.充分混勻?qū)ΡＷC反應(yīng)結(jié)果的準(zhǔn)性很重要，在加液后請輕輕叩擊邊緣以保證混勻。

8.室溫反應(yīng)，請嚴(yán)格控制在25～28℃。

9.洗滌過程是至關(guān)重要的，洗滌不充分會使精確度下降并導(dǎo)致結(jié)果誤差較大。

10.試驗中標(biāo)準(zhǔn)品和樣本檢測時建議作雙復(fù)孔。

11.加樣過程中避免氣泡的產(chǎn)生。

12.血清和血漿標(biāo)本的檢測時，檢測抗體的孵育時間應(yīng)適當(dāng)延長。

檢測前準(zhǔn)備工作:

1.試劑盒自冰箱中取出后應(yīng)置室溫（25～28℃）平衡20分鐘；每次檢測后剩余試劑請及時于2～8℃保存。

2.將濃縮洗滌液用雙蒸水或去離子水稀釋(1份加19份水)。

3.如有5X準(zhǔn)品稀釋液，請按所需量用雙蒸水或去離子水稀釋(1份加4水)。

4.標(biāo)準(zhǔn)品: 按標(biāo)簽復(fù)溶體積加入標(biāo)準(zhǔn)品稀釋液復(fù)溶使 CRP 終濃度達到 10000pg/ml，室溫反應(yīng)，請嚴(yán)格控制在 25～28℃，靜置 10~15 分鐘后

輕輕混懸（建議抽吸幾次）待徹底溶解，用標(biāo)準(zhǔn)品稀釋液倍比梯度稀釋后依次加入檢測孔中。（標(biāo)準(zhǔn)曲線取七個點，最高濃度為 10000pg/ml,

標(biāo)準(zhǔn)品稀釋液直接加入作為 0 濃度.）

洗滌方法:

自動洗板機或人工洗板：每孔洗滌液為300ul，注入與吸出間隔15-30秒。洗板5次。最后一次洗板完成后將板倒扣著在厚吸水紙上用力拍干。

實驗過程需自備的材料：

1.不同規(guī)格的加樣槍及相應(yīng)的槍頭；

2.酶標(biāo)儀；

3.自動洗板機；

4.去離子水或雙蒸水；

操作步驟:

1.通過計算并確定一次性實驗所需的板條數(shù)，取出所需板條放置在框架內(nèi)，暫時用不到板條請放回鋁箔袋密封，保存于4℃。

2.建議設(shè)置本底較正孔，即空白孔,設(shè)置方法為該孔只加TMB顯色液和中止液。每次實驗均需做標(biāo)準(zhǔn)品對照并畫出標(biāo)準(zhǔn)曲線。

上海傳秋生物科技有限公司 424100.com.cn人的CRP參考標(biāo)準(zhǔn)曲線

0

0.5

1

1.5

2

2.5

3

0 312.5 625 1250 2500 5000 10000

人的CRP濃度（pg/ml）

O

D值

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3.分別將標(biāo)本或不同濃度標(biāo)準(zhǔn)品(100ul/孔)加入相應(yīng)孔中，用封板膠紙封住反應(yīng)孔，室溫（25～28℃）孵育120分鐘。如果是血清血漿樣本，

不同樣本稀釋比例不一樣，一般范圍在20~450倍，如無明確范圍，建議從100倍稀釋，如果樣本濃度過高，超過檢測范圍，請加大稀釋倍數(shù)

后重新稀釋檢測。

4.洗板5次，且最后一次置厚吸水紙上拍干。

5.加入生物素連接抗體工作液(100ul/孔)。用封板膠紙封住反應(yīng)孔，室溫（25～28℃）孵育60分鐘。

6.洗板5次，且最后一次置厚吸水紙上拍干。

7. 加入親和素連接HRP工作液(100ul/孔)。用封板膠紙封住反應(yīng)孔，室溫（25～28℃）孵育20分鐘。

8. 洗板5次，且最后一次置厚吸水紙上拍干。

9. 加入顯色劑TMB100ul/孔，避光室溫（25～28℃）孵育20分鐘。

10. 加入中止液50ul/孔,混勻后即刻測量OD450值。

結(jié)果判斷:

1.復(fù)孔的值在20%的差異范圍內(nèi)結(jié)果才有效，復(fù)孔的值平均后可作為測量值。

2.每個標(biāo)準(zhǔn)品或標(biāo)本的OD值應(yīng)減去本底校正孔的OD值。

3.手工繪制標(biāo)準(zhǔn)曲線。以標(biāo)準(zhǔn)品濃度作橫坐標(biāo)，OD值作縱坐標(biāo)，以平滑線連接各標(biāo)準(zhǔn)品的坐標(biāo)點。通過標(biāo)本的OD值可在標(biāo)準(zhǔn)曲線上查出其

濃度。

4.若標(biāo)本 OD 值高于標(biāo)準(zhǔn)曲線上限，應(yīng)適當(dāng)稀釋后重測，計算濃度時應(yīng)乘以稀釋倍數(shù)。

典型數(shù)值和參考曲線

濃度pg/ml 典型OD值1 典型OD值2 OD平均值

0 0.0845 0.1143 0.0994

312.5 0.2661 0.2951 0.2806

625 0.4296 0.467 0.4483

1250 0.7635 0.7835 0.7735

2500 1.2203 1.2399 1.2301

5000 1.7987 1.8315 1.8151

10000 2.7075 2.6207 2.6641

人高敏CRP參考標(biāo)準(zhǔn)曲線

注意：本圖僅供參考，應(yīng)以同次試驗標(biāo)準(zhǔn)品所繪標(biāo)準(zhǔn)曲線計算標(biāo)本含量。

靈敏度，特異性和重復(fù)性:

1.靈敏度：多次重復(fù)結(jié)果表明，最小檢出量為18.4pg/ml。

2. 重復(fù)性：板內(nèi)，板間變異系數(shù)均<10%.參考文獻:

1. Johnson HL et al; Applications of acute phase reactants in infectious diseases J.Microbiol. Immunol. Infect. 1999, 32: 73

2. Helgeson N et al; C - Reactive Protein : Laboratory Medicine, Vol. 2 (Race G. J., Ed.),Harper & Row, Hagerstown, chapter 29 (1973).

3. Gewurz H et al; C-Reactive Protein and the Acute Phase Response. Adv in Int Med, 1982,27: 345

4. Frank, R. and R. Hargreaves (2003) Nat. Rev. Drug Disc. 2:566.

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ELISA Kit for the Quantitative Analysis of Human high sensitive CRP

The human CRP ELISA (enzyme-linked immunosorbent assay) kit is used for detection of human CRP in cell culture

supernatants,human serum and plasma.THE ELISA KIT IS FOR RESEARCH USE ONLY. Please read this instruction manual carefully

and check out the material provided before use, and you can contact with our company if any questions. You can enter our website or call

us for other aim.

Introduction

C-Reactive Protein (CRP) is the prototypical acute phase protein in humans and is an important mediator of immune host defense (1,

2) of the pentraxin family (4-6).. Human CRP precursor is a 224 amino acids protein.The mature human CRP protein has 206 amino

acids that are noncovalently linked to form the homo pentameric ring structure .

Serum concentration of CRP increases significantly in cases of both infectious and noninfectious inflammation, of tissue damage and

necrosis and in the presence of malignant tumors. In response to infection, inflammation or tissue damage, the level of CRP in human

serum can increase 1,000 fold within 24~48 hours.

CRP exhibits Ca2+ dependent binding to ligands. Phosphocholine (PCh), a constituent of many bacterial and fungal walls, is a

principal ligand of CRP. CRP also binds to the membrane of injured cells, membrane and nuclear components of necrotic and apoptotic

cells. Upon binding with the ligands, CRP is recognized by C1q and initiates the activation of complement cascade. Ligand bound CRP

also binds to Fcγ RI and Fcγ RIIa on phagocytes and activates phogocytotic responses.CRP produced exclusively in the liver.

Interleukin-6 is the mediator for the synthesis by the hepatocytes . IL-6,IL-1 and glucocorticoids are the major inducer of the CRP gene.

Principles of the Test

The kits is a solid sandwich enzyme-linked immunosorbent assay for detection of human CRP. An anti-human CRP monoclonal

antibody has been absorbed onto the wells of the microtiter strips provided. Samples including specimens or standards were pipetted into

wells. The human CRP in specimens or standards would be captured by the coated antibody and the free others were removed by

washing. The human CRP biotin-conjugated antibody were added and binds to human CRP captured by the first antibody, which formed

a sandwich. Streptavidin-HRP would be added and binds to the biotin conjugated antibody, then free Streptavidin-HRP would be

removed during a wash step. After this, subtrate solution would be added and catalyzed by the HRP, and a coloured product is formed.

The intensity of the colored product is used to calculate in proportion to the amount of human CRP in the original.

Materials provided with the kits:

reagent 96/48Test Kit

Human high sensitive CRP Antibody-Coated Wells 12 strips/6 strips

Standard Diluent 10 ml/5ml

Human high sensitive CRP Standard 4/2vial(s)

Human high sensitive CRP biotinylated Antibody 10ml/5ml

Streptavidin conjugated HRP 10ml/5ml

Wash Buffer Concentrate 20× 30ml/15ml

TMB 10ml/5 ml

Stop Solution 5ml/3 ml

Plate Covers 3/2

Complete Instruction Manual 1Specimen Collection

1.Collecting specimen as following:

A.The particulate of the cell culture supernatants should be removed before use.

B.Serum was obtained from clot at room temperature.

C.Please collect plasma with EDTA.

D.Assay immediately or store samples at －20℃. Avoid free-thaw cycles.

2.Antiseptic and anticoagulant should not appear in Serum samples.

3.Any particulate should be removed from samples before use.

4. Do not use grossly hemolyzed or lipemic samples.

Note: Strongly recommend that the serum and plasma samples should be diluent as doubling dilution before use.

Precautions for use:

1.Please storage the Kit at 2～8℃。

2. Washing buffer concentrate may have crystal in low temperature, and you can melt its in water-bath before use.

3. Please discard the dissolved standard after 3 days for use.

4. Avoid contact of substrate solution with oxidizing agents and metal.

5. Usage of disposable pipette tips avoid microbial contamination or cross-contamination of reagents or specimens.

6. Do not mix or substitute reagents with those from other lots or other sources.

7. To ensure the adequate mixure of added reagents, please tap gently the plate after the wells were filled with liquid.

8. Incubation temperature should be 25～28℃.

9. Wash step was crucial for whole assay process.

10. Duplicate wells of the same sample were recommended in assay process.

11. Avoid the foam while pour the liquid into wells.

12. For serum or plasma samples ,the biotin-conjugated antibody should be incubate for at least 90 minutes.

Reagent Preparation

1.The reagents should be warmed up to room temperature before use. The remanent reagents must reseal and put into refrigeratory

again as soon as possible.

2. Dilute 1ml of wash buffer Concentrate into 19ml deionized or distilled water to work.

3. If you have a 5x standard diluent, please dilute it with double steaming water or deionized water.

4. Add standard dilution solution to the bottle according to the volume of the label and wait15 minutes for complete dissolution. And in turn

add the half concentration diluent by standard diluent .

Wash step:

Automated microplate washer or operating by pipette: Each well should be pour into300ul wash buffer and soak 15 or 30 seconds,then

be aspirated, five times process were repeated. After the last wash, remove remaining wash buffer by aspirating.Invert the plate and blot

it against clean paper towels.

Materials Required But Not Provided

1. pipettes and pipette tips

2. Microwell strip reader capable of reading at 450 nm (540 nm as optional reference wave length)

3. automated microplate washer

4.Glass-distilled or deionized water

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Assay procedure

1.The needed strips were putted into the frame, the remains were returned into foil pouch and resealed.

2.Blank well were recommended, which only color reagent and stop solution be added. It is suggested that each testing with gradient

density of standard for standard curve.

3.Add 100ul of standard or sample. then cover with the Plate Covers provided.Incubate for 120 minutes at room temperature. If is plasma

serum samples, different sample dilution ratio is different, generally range in 20 ~ 450 times, if there is no definite scope, advice from 100

times dilution, if the sample concentration is too high, more than test scope, please increase after diluted times dilution test again。4.Five

times wash process were repeated.

5.Add 100ul of detetion antibody. Cover with the Plate Covers provided.Incubate for 1 hour at room temperature.

6.Five times wash process were repeated.

7.Add 100ul of Streptavidin-HRP. Cover with the Plate Covers provided. Lucifugal incubation for 20 minutes at room temperature.

8.Five times wash process were repeated.

9.Add 100ul of TMB，Lucifugal incubation for 20 minutes at room temperature.

10.Add 50ul of stop solution to each well, determine the optica density of each well within 10 minutes.

Calculation of Results

1.Duplicates should be within 20 per cent of the mean. Average absorbance values for each set of duplicate samples were used as

detection results.

2.The blank absorbance values of subtract should be deducted.

3.Drawing a best fit curve through the points of graph. Draw the standard curve by plotting assayed OD valure (on the Y axis) vs.

concentration (on the X axis). The sample concentration was obtained based on its OD value founding in the standard concentration

curve.

4.If the values obtained are not within the expected range of the standard, Samples should be dilute and assay again.

Typical Data and Standard Curve

concentration

(pg/ml)

Typical data 1 Typical data 2 Average

0 0.0845 0.1143 0.0994

31.25 0.2661 0.2951 0.2806

62.5 0.4296 0.467 0.4483

125 0.7635 0.7835 0.7735

250 1.2203 1.2399 1.2301

500 1.7987 1.8315 1.8151

10000 2.7075 2.6207 2.6641

Human high sensitive CRP standard curveHuman high sensitive CRP Standard Curve

Sensitivity, Specificity, Repeatability

Sensitivity: repeated assays were evaluated and the minimum detectable dose was 18. 4pg/ml.

Repeatability: The coefficient of variation between wells or plates is less than 10 per cent.

REFERENCES:

1. Johnson HL et al; Applications of acute phase reactants in infectious diseases J.

Microbiol. Immunol. Infect. 1999, 32: 73

2. Helgeson N et al; C - Reactive Protein : Laboratory Medicine, Vol. 2 (Race G. J., Ed.),Harper & Row, Hagerstown, chapter 29 (1973).

3. Gewurz H et al; C-Reactive Protein and the Acute Phase Response. Adv in Int Med, 1982,27: 345

4. Frank, R. and R. Hargreaves (2003) Nat. Rev. Drug Disc. 2:566.

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