上海传秋生物科技有限公司 www.chuanqiubio.com

人的正常 T 细胞表达和分泌因子定量分析酶联免疫检测试剂盒

本试剂盒仅供科研使用。用于体外定量检测人血清、血浆或细胞培养上清液中的 CCL5 浓度。使用前请仔细阅读说明书并检查试剂组分

是否完整, 如有疑问请与安徽巧伊生物科技有限公司联系，我们将提供力所能及的帮助。

CCL5 简介：

RANTES，也叫 CCL5，是一个分子量为 8kDa 的单体蛋白，是属于 CC 趋化因子家族的细胞因子。CCL5 前体蛋白是一个 91 个氨基酸的多肽，

带有 23 个氨基酸的信号肽，经过裂解后产生 68 个氨基酸的成熟蛋白。人的 CCL5 与小鼠的 CCL5 的蛋白同源性大概是 85%。

人的 CCL5 基因位于 17 号染色体上。据报道，CCL5 蛋白可由 T 细胞，成纤维细胞和血小板细胞等产生。炎症反应中通过激发白细胞趋

向炎症部位，人的 CCL5 蛋白在炎症反应中起重要作用。CCL5 是 T 细胞，单核细胞，树枝状细胞的化学诱导剂，能够刺激 NK 细胞的增殖和

细胞毒性。CCL5 也在某些肿瘤的发生和转移中起作用，在一些恶性肿瘤中 CCL5 也会持续高表达。

检测原理:

本试剂盒采用双抗体夹心ELISA法检测样本中CCL5的浓度。CCL5捕获抗体已预包被于酶标板上，当加入标本或参考品时，其中的CCL5会

与捕获抗体结合，其它游离的成分通过洗涤的过程被除去。当加入生物素化的抗人CCL5抗体后，抗人CCL5抗体与CCL5接合，形成夹心的免

疫复合物，其它游离的成分通过洗涤的过程被除去。随后加入辣根过氧化物酶标记的亲合素。生物素与亲合素特异性结合，亲合素连接的

酶就会与夹心的免疫复合物连接起来；其它游离的成分通过洗涤的过程被除去。最后加入显色剂，若样本中存在CCL5将会形成免疫复合物，

辣根过氧化物酶会催化无色的显色剂氧化成蓝色物质，在加入终止液后呈黄色。通过酶标仪检测，读其450nm处的OD值，CCL5浓度与OD450值

之间呈正比，通过参考品绘制标准曲线，对照未知样本中OD值，即可算出标本中CCL5浓度。

人CCL5定量分析酶联免疫检测试剂盒组成:

组分 规格（96T/48T）

人CCL5预包被板 12条/6条

标准品稀释液 20ml/10ml

人CCL5标准品 2支/1支(冻干)

人CCL5生物素化抗体 10ml/5ml

亲和素连接的HRP酶 10ml/5ml

浓缩洗涤液 20× 30ml/15ml

TMB底物 10ml/5ml

中止液 5ml/3ml

封板胶纸 3/2张

说明书 1份

标本收集:

1.标本的收集请按下列流程进行操作；

A.细胞上清标本离心去除悬浮物后即可；

B.血清标本应是自然凝固后，取上清，避免在冰箱中凝固血液；

C.血浆标本，推荐用EDTA的方法收集

D.若待测样本不能及时检测，标本收集后请分装，冻存于－20℃，避免反复冻融。

2.血清标本不应添加任何防腐剂或抗凝剂；

3.标本应清澈透明，检测前样本中如有悬浮物应通过离心去除。

4.请勿使用溶血，高血脂或污染的标本检测，否则结果将不准确。上海传秋生物科技有限公司 www.chuanqiubio.com

注意事项:

1.试剂盒请保存在2～8℃。

2.浓缩洗涤液因在低温下可能有结晶，请水浴加热使结晶完全溶解后再配制工作液。

3.标准品复溶加样后，剩余部份请丢弃。

4.底物请勿接触氧化剂和金属。

5.加样时，请及时更换枪头，避免交叉污染。

6.严禁混用不同批号的试剂盒组份。

7.充分混匀对保证反应结果的准性很重要，在加液后请轻轻叩击边缘以保证混匀。

8.室温反应，请严格控制在25～28℃。

9.洗涤过程是至关重要的，洗涤不充分会使精确度下降并导致结果误差较大。

10.试验中标准品和样本检测时建议作双复孔。

11.加样过程中避免气泡的产生。

12.血清和血浆标本的检测时，检测抗体的孵育时间应适当延长。

检测前准备工作:

1.试剂盒自冰箱中取出后应置室温（25-28℃）平衡20分钟；每次检测后剩余试剂请及时于2～8℃保存。

2.将浓缩洗涤液用双蒸水或去离子水稀释(1份加19份水)。

3. 将5×标准品稀释液用双蒸水或去离子水稀释(1份加4份水)。

4.标准品: 按标签复溶体积加入标准品稀释液复溶使CCL5终浓度达到1000pg/ml，室温反应，请严格控制在25～28℃，静置15~20分钟后轻

轻混悬（建议抽吸几次）待彻底溶解，用标准品稀释液按如下方法在干净的玻璃管或离心管中稀释后依次加入检测孔中。（标准曲线取八

个点，最高浓度为1000 pg/ml,不需稀释，标准品稀释液直接加入作为0浓度.）

稀释浓度（pg/ml） 750 500 375 250 125 62.5

高浓度标准品（1000 pg/ml）加入量（μ l） 180 120 90 60 30 15

1×标准品稀释液加入量（μ l） 60 120 150 180 210 225

稀释得到相应浓度的总体积（μ l） 240 240 240 240 240 240

洗涤方法:

自动洗板机或人工洗板：每孔洗涤液为300ul，注入与吸出间隔15-30秒。洗板5次。最后一次洗板完成后将板倒扣着在厚吸水纸上用力拍干。

实验过程需自备的材料：

1.不同规格的加样枪及相应的枪头；

2.酶标仪；

3.自动洗板机；

4.去离子水或双蒸水；

操作步骤:

1.通过计算并确定一次性实验所需的板条数，取出所需板条放置在框架内，暂时用不到板条请放回铝箔袋密封，保存于 4℃。

2.建议设置本底较正孔，即空白孔,设置方法为该孔只加 TMB 显色液和中止液。每次实验均需做标准品对照并画出标准曲线。

3.分别将标本或不同浓度标准品(100ul/孔)加入相应孔中，用封板胶纸封住反应孔，室温（25-28℃）孵育120分钟对于血清样本的稀释倍

数一般4~100倍稀释，如无准确稀释范围，从2倍开始稀释。细胞上清需根据实验结果，如果超过试剂盒的检测限，请相应稀释后再检测。

4.洗板 5 次，且最后一次置厚吸水纸上拍干。

5.加入生物素化抗体工作液(100ul/孔)。用封板胶纸封住反应孔，室温（25-28℃）孵育60分钟。

6.洗板5次，且最后一次置厚吸水纸上拍干。

7.加入亲和素连接的HRP酶工作液(100ul/孔)。用封板胶纸封住反应孔，避光室温（25-28℃）孵育20分钟。

8.洗板5次，且最后一次置厚吸水纸上拍干。

9.加入显色剂TMB100ul/孔，避光室温（25-28℃）孵育4~6，最好5分钟。因为反应速度非常快，此步骤建议用排枪加入TMB，否则误

会非常大。

10.加入中止液50ul/孔,混匀后即刻测量OD450值。人CCL5参考标准曲线

0

0.5

1

1.5

2

2.5

3

0 62.5 125 250 375 500 750 1000

人CCL5的浓度(pg/ml)

O

D值

上海传秋生物科技有限公司 www.chuanqiubio.com

结果判断:

1.复孔的值在20%的差异范围内结果才有效，复孔的值平均后可作为测量值。

2.每个标准品或标本的OD值应减去本底校正孔的OD值。

3.手工绘制标准曲线。以标准品浓度作横坐标，OD值作纵坐标，以平滑线连接各标准品的坐标点。通过标本的OD值可在标准曲线上查出

其浓度。

4.若标本OD值高于标准曲线上限，应适当稀释后重测，计算浓度时应乘以稀释倍数。

典型数值和参考曲线

浓度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 参考标准曲线

注意：本图仅供参考，应以同次试验标准品所绘标准曲线计算标本含量。

灵敏度，特异性和重复性:

1.灵敏度：多次重复结果表明，最小检出量为5.9pg/ml。

2.特异性：与人的ANG 、EGF 、Epo 、FGF acidic 、HGF、 IFN-γ 、 FGF basic 及小鼠的CCL5无交叉反应性。

3.重复性：板内，板间变异系数均<10%.

参考文献:

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. 上海传秋生物科技有限公司 www.chuanqiubio.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.上海传秋生物科技有限公司 www.chuanqiubio.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 evaluated 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.