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人 C 反應蛋白（超敏）定量分析酶聯免疫檢測試劑盒

本試劑盒僅供科研使用 。用於體外定量檢測人血清 、血漿或細胞培養上清液中的 CRP 濃度 。使用前請仔細閱讀說明書並檢查試劑組分

是否完整 。如有產品包裝破損或質量投拆 ，請在收到貨一個月之內聯係尊龍凱時 。

CRP 簡介 ：

C 反應蛋白（CRP）是人體內的急性反應蛋白 ，在人體免疫防禦反應中重要的介導物 ，屬於穿透素家族的蛋白 。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 ，通過非共價結合的方式形成五聚體的環狀結構 。

無論是感染還是非感染性的炎症 、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 ，炎症反應及組織壞死

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

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

調亡的細胞等結合 。一旦與受體結合，CRP 蛋白就會啟動 C1q ，從而引起一係列的級聯反應 。CRP 蛋白也會與吞噬細胞的表麵 Fcγ RI 和 Fcγ

RII 結合 ，從而啟動吞噬細胞的吞噬反應 。

CRP 主要在肝內產生 ，IL-6 是肝實質細胞產生 CRP 蛋白的主要介導物 ，此外 IL-1 和糖皮質激素也是 CRP 蛋白產生重要的誘導物 。

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捕獲抗體已預包被於酶標板上 ，當加入標本或參考品時 ，其中的CRP會與

捕獲抗體結合 ，其它遊離的成分通過洗滌的過程被除去 。當加入與生物素化的抗人CRP抗體後 ，抗人CRP抗體與CRP接合 ，形成夾心的免疫複

合物 ，其它遊離的成分通過洗滌的過程被除去 。再加入親和素連接的HRP結合物 ，生物素與親和素結合 ，其它遊離成分通過洗滌的過程被除

去 ，最後加入顯色劑 ，若樣本中存在CRP將會形成免疫複合物 ，辣根過氧化物酶會催化無色的顯色劑氧化成藍色物質 ，在加入終止液後呈黃

色 。通過酶標儀檢測 ，讀其450nm處的OD值 ，CRP濃度與OD450值之間呈正比 ，通過參考品繪製標準曲線 ，對照未知樣本中OD值 ，即可算出標

本中CRP濃度 。

人高敏CRP定量分析酶聯免疫檢測試劑盒組成:

組分 規格（96T/48T）

人高敏CRP預包被板 12條/6條

標準品稀釋液 10ml/5ml

人高敏CRP標準品 4支/2支(凍幹)

生物素化的抗CRP抗體 10ml/5ml

親和素連接的HRP結合物 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

標本收集:

1.標本的收集請按下列流程進行操作 ；

A.細胞上清標本離心去除懸浮物後即可 ；

B.血清標本應是自然凝固後 ，取上清 ，避免在冰箱中凝固血液 ；

C.血漿標本 ，推薦用EDTA的方法收集若待測樣本不能及時檢測 ，

D.標本收集後請分裝 ，凍存於－20℃ ，避免反複凍融 。2.血清標本不應添加任何防腐劑或抗凝劑 ；

3.標本應清澈透明 ，檢測前樣本中如有懸浮物應通過離心去除 。

4.請勿使用溶血 ，高血脂或汙染的標本檢測 ，否則結果將不準確 。

注 ：人血清或血漿樣本請用標準品稀釋液做稀釋後再檢測 ，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晶 ，請水浴加熱使結晶完全溶解後再配製工作液。

3.標準品複溶加樣後 ，剩餘部分請丟棄 。

4.底物請勿接觸氧化劑和金屬 。

5.加樣時 ，請及時更換槍頭 ，避免交叉汙染 。

6.嚴禁混用不同批號的試劑盒組份 。

7.充分混勻對保證反應結果的準性很重要 ，在加液後請輕輕叩擊邊緣以保證混勻 。

8.室溫反應 ，請嚴格控製在25～28℃ 。

9.洗滌過程是至關重要的 ，洗滌不充分會使精確度下降並導致結果誤差較大 。

10.試驗中標準品和樣本檢測時建議作雙複孔 。

11.加樣過程中避免氣泡的產生 。

12.血清和血漿標本的檢測時 ，檢測抗體的孵育時間應適當延長 。

檢測前準備工作:

1.試劑盒自冰箱中取出後應置室溫（25～28℃）平衡20分鍾 ；每次檢測後剩餘試劑請及時於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.標準品: 按標簽複溶體積加入標準品稀釋液複溶使 CRP 終濃度達到 10000pg/ml ，室溫反應 ，請嚴格控製在 25～28℃ ，靜置 10~15 分鍾後

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

標準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 。最後一次洗板完成後將板倒扣著在厚吸水紙上用力拍幹 。

實驗過程需自備的材料 ：

1.不同規格的加樣槍及相應的槍頭 ；

2.酶標儀 ；

3.自動洗板機 ；

4.去離子水或雙蒸水 ；

操作步驟:

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

2.建議設置本底較正孔 ，即空白孔,設置方法為該孔隻加TMB顯色液和中止液 。每次實驗均需做標準品對照並畫出標準曲線 。

上海尊龍凱時生物科技有限公司 www.ruianjituan.com人的CRP參考標準曲線

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.分別將標本或不同濃度標準品(100ul/孔)加入相應孔中 ，用封板膠紙封住反應孔 ，室溫（25～28℃）孵育120分鍾 。如果是血清血漿樣本 ，

不同樣本稀釋比例不一樣 ，一般範圍在20~450倍 ，如無明確範圍 ，建議從100倍稀釋 ，如果樣本濃度過高 ，超過檢測範圍 ，請加大稀釋倍數

後重新稀釋檢測 。

4.洗板5次 ，且最後一次置厚吸水紙上拍幹 。

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

6.洗板5次 ，且最後一次置厚吸水紙上拍幹 。

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

8. 洗板5次 ，且最後一次置厚吸水紙上拍幹 。

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

10. 加入中止液50ul/孔,混勻後即刻測量OD450值 。

結果判斷:

1.複孔的值在20%的差異範圍內結果才有效，複孔的值平均後可作為測量值 。

2.每個標準品或標本的OD值應減去本底校正孔的OD值 。

3.手工繪製標準曲線 。以標準品濃度作橫坐標 ，OD值作縱坐標 ，以平滑線連接各標準品的坐標點 。通過標本的OD值可在標準曲線上查出其

濃度 。

4.若標本 OD 值高於標準曲線上限 ，應適當稀釋後重測 ，計算濃度時應乘以稀釋倍數 。

典型數值和參考曲線

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參考標準曲線

注意 ：本圖僅供參考 ，應以同次試驗標準品所繪標準曲線計算標本含量 。

靈敏度 ，特異性和重複性:

1.靈敏度 ：多次重複結果表明 ，最小檢出量為18.4pg/ml 。

2. 重複性 ：板內 ，板間變異係數均<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 evalsuated 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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