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人的正常 T 細胞表達和分泌因子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 CCL5 濃度 。使用前請仔細閱讀說明書並檢查試劑組分

是否完整, 如有疑問請與安徽巧伊生物科技有限公司聯係 ，尊龍凱時將提供力所能及的幫助 。

CCL5 簡介 ：

RANTES ，也叫 CCL5 ，是一個分子量為 8kDa 的單體蛋白 ，是屬於 CC 趨化因子家族的細胞因子 。CCL5 前體蛋白是一個 91 個氨基酸的多肽 ，

帶有 23 個氨基酸的信號肽 ，經過裂解後產生 68 個氨基酸的成熟蛋白 。人的 CCL5 與小鼠的 CCL5 的蛋白同源性大概是 85% 。

人的 CCL5 基因位於 17 號染色體上 。據報道 ，CCL5 蛋白可由 T 細胞 ，成纖維細胞和血小板細胞等產生 。炎症反應中通過激發白細胞趨

向炎症部位 ，人的 CCL5 蛋白在炎症反應中起重要作用 。CCL5 是 T 細胞 ，單核細胞，樹枝狀細胞的化學誘導劑 ，能夠刺激 NK 細胞的增殖和

細胞毒性 。CCL5 也在某些腫瘤的發生和轉移中起作用 ，在一些惡性腫瘤中 CCL5 也會持續高表達 。

檢測原理:

本試劑盒采用雙抗體夾心ELISA法檢測樣本中CCL5的濃度 。CCL5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CCL5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 。當加入生物素化的抗人CCL5抗體後 ，抗人CCL5抗體與CCL5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 。隨後加入辣根過氧化物酶標記的親合素 。生物素與親合素特異性結合 ，親合素連接的

酶就會與夾心的免疫複合物連接起來 ；其它遊離的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CCL5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 ，CCL5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CCL5濃度 。

人CCL5定量分析酶聯免疫檢測試劑盒組成:

組分 規格（96T/48T）

人CCL5預包被板 12條/6條

標準品稀釋液 20ml/10ml

人CCL5標準品 2支/1支(凍幹)

人CCL5生物素化抗體 10ml/5ml

親和素連接的HRP酶 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

D.若待測樣本不能及時檢測 ，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 。上海尊龍凱時生物科技有限公司 www.ruianjituan.com

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 ，剩餘部份請丟棄 。

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. 將5×標準品稀釋液用雙蒸水或去離子水稀釋(1份加4份水) 。

4.標準品: 按標簽複溶體積加入標準品稀釋液複溶使CCL5終濃度達到1000pg/ml ，室溫反應 ，請嚴格控製在25～28℃ ，靜置15~20分鍾後輕

輕混懸（建議抽吸幾次）待徹底溶解 ，用標準品稀釋液按如下方法在幹淨的玻璃管或離心管中稀釋後依次加入檢測孔中 。（標準曲線取八

個點 ，最高濃度為1000 pg/ml,不需稀釋 ，標準品稀釋液直接加入作為0濃度.）

稀釋濃度（pg/ml） 750 500 375 250 125 62.5

高濃度標準品（1000 pg/ml）加入量（μ l） 180 120 90 60 30 15

1×標準品稀釋液加入量（μ l） 60 120 150 180 210 225

稀釋得到相應濃度的總體積（μ l） 240 240 240 240 240 240

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 。

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分鍾對於血清樣本的稀釋倍

數一般4~100倍稀釋 ，如無準確稀釋範圍 ，從2倍開始稀釋 。細胞上清需根據實驗結果 ，如果超過試劑盒的檢測限 ，請相應稀釋後再檢測 。

4.洗板 5 次 ，且最後一次置厚吸水紙上拍幹 。

5.加入生物素化抗體工作液(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.加入親和素連接的HRP酶工作液(100ul/孔) 。用封板膠紙封住反應孔 ，避光室溫（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4~6 ，最好5分鍾 。因為反應速度非常快 ，此步驟建議用排槍加入TMB ，否則誤

會非常大 。

10.加入中止液50ul/孔,混勻後即刻測量OD450值 。人CCL5參考標準曲線

0

0.5

1

1.5

2

2.5

3

0 62.5 125 250 375 500 750 1000

人CCL5的濃度(pg/ml)

O

D值

上海尊龍凱時生物科技有限公司 www.ruianjituan.com

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 0.115 0.103 0.109

62.5 0.289 0.121 0.205

125 0.467 0.263 0.365

250 0.636 0.56 0.598

375 0.933 0.849 0.891

500 1.317 1.261 1.289

750 1.861 1.649 1.755

1000 2.613 2.229 2.421

人CCL5 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5.9pg/ml 。

2.特異性 ：與人的ANG  、EGF  、Epo  、FGF acidic  、HGF 、 IFN-γ  、 FGF basic 及小鼠的CCL5無交叉反應性 。

3.重複性 ：板內 ，板間變異係數均<10%.

參考文獻:

1. Schall, T.J. (1991) Cytokine 3:165.

2. Graham, G.J. et al. (1990) Nature 344:442.

3. Neote, K. et al. (1993) Cell 72:415.

4. Oppenheim, J.J. et al. (1991) Annu. Rev. Immunol. 9:617.

5. Murphy, P.M. (1994) Annu. Rev. Immunol. 12:593.

6. Schall, T.J. et al. (1993) J. Exp. Med. 177:1821. 上海尊龍凱時生物科技有限公司 www.ruianjituan.com

ELISA Kit for the Quantitative Analysis of Human CCL5

The human CCL5 ELISA (enzyme-linked immunosorbent assay) kit is used for detection of human CCL5 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

RANTES, also known as CCL5, is an 8 kDa monomeric protein that belongs to the CC- family of chemokines. RANTES precursor is a

91 amino acid (aa) residue polypeptide with a 23 aa hydrophobic signal peptide that is cleaved to generate the 68 aa mature

protein. Mature human CCL5 shares85% aa sequence identity with mouse CCL5 .

CCL5 has been localized to chromosome 17 in humans. It is reported to be produced by fibroblasts, platelets, and T cells. RANTES

plays an important role in the immune response by initiating the recruitment of leukocytes to the site of inflammation. It is a

chemoattractant for T cells, monocytes, and dendritic cells, and may increase the cytotoxicity and promote proliferation of NK cells.

RANTES is also involved in the progression and metastasis of some forms of cancer, and constitutive expression has been demonstrated

in some malignant tumors.

Principles of the Test

The kits is a solid sandwich enzyme-linked immunosorbent assay for detection of human CCL5. An anti-human CCL5 monoclonal

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

wells. The human CCL5 in specimens or standards would be captured by the coated antibody and the free others were removed by

washing. The human CCL5 biotin-conjugated antibody were added and binds to human CCL5 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 CCL5 in the original specimen.

Materials provided with the kits:

Reagent 96/48Test Kit

Human CCL5 Antibody-Coated Wells 12 strips/6 strips

Standard Diluent 20 ml/10ml

Human CCL5 Standard 2/1vial(s)

Human CCL5 Detetion Antibody 10ml/5ml

Streptavidin-HRP 10ml/5ml

Wash Buffer Concentrate 20× 30ml/15ml

TMB 10ml/5ml

Stop Solution 5ml/3ml

Plate Covers 3/2

Complete Instruction Manual 1

Specimen 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.上海尊龍凱時生物科技有限公司 www.ruianjituan.com

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 remains after use of the dissolved standard.

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. Dilute 1ml of 5×sample diluent into 4ml deionized or distilled water to 1×sample diluent.

4. Add the standard dilution solution to the bottle according to the volume of the label and wait 15 minutes for complete dissolution.

Incubation temperature should be 25～28℃,this is highest concentrations(1000 pg/ml) in Standard curves,and 1×sample diluent added

the well as zero concentrations point, Then diluent along with flowing:

Concentrations after diluenting（pg/ml） 750 500 375 250 125 62.5

highest concentrations（1000 pg/ml）added（μ l） 180 120 90 60 30 15

1×sample diluent added（μ l） 60 120 150 180 210 225

The volume of diluenting standard(μ l) 240 240 240 240 240 240

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 asoptional reference wave length)

3. automated microplate washer

4.Glass-distilled or deionized water

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.Cover with the Plate Covers provided.Incubate for 2 hours at room temperature. The suggested

dilution for normal serum/plasma is 4~100 fold. .If the OD value of sample beyond the Kit scope ,you should re-assay after diluent the

sample.

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 5 minutes at room temperature.

10.Add 50ul of stop solution to each well, determine the optical

Calculation of Results

1.Duplicates should be within 20 percent 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 0.115 0.103 0.109

62.5 0.289 0.121 0.205

125 0.467 0.263 0.365

250 0.636 0.56 0.598

375 0.933 0.849 0.891

500 1.317 1.261 1.289

750 1.861 1.649 1.755

1000 2.613 2.229 2.421

Human CCL5 Standard Curve

Sensitivity, Specificity, Repeatability

Sensitivity: repeated assays were evalsuated and the minimum detectable dose was 5.9 pg/ml.

Specificity : No significant cross-reactivity or interference with human ANG ,EGF ,Epo ,FGF acidic ,HGF, IFN-γ , FGF basic and Mouse

CCL5.

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

REFERENCES:

1. Schall, T.J. (1991) Cytokine 3:165.

2. Graham, G.J. et al. (1990) Nature 344:442.

3. Neote, K. et al. (1993) Cell 72:415.

4. Oppenheim, J.J. et al. (1991) Annu. Rev. Immunol. 9:617.

5. Murphy, P.M. (1994) Annu. Rev. Immunol. 12:593.

6. Schall, T.J. et al. (1993) J. Exp. Med. 177:1821.