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大鼠单核细胞趋化蛋白 1 定量分析酶联免疫检测试剂盒
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大鼠单核细胞趋化蛋白 1 定量分析酶联免疫检测试剂盒

本试剂盒仅供科研使用。用于体外定量检测大鼠血清、血浆或细胞培养上清液中的 MCP-1 浓度。使用前请仔细阅读说明书并检查试剂

组分是否完整,如有产品包装破损或质量投拆,请在收到货一个月之内联系我们。

MCP-1 简介:

单核细胞趋化蛋白 1(MCP-1/CCL2),也被称为单核细胞趋化与激活因子(MCAF),是单核细胞和 T 細胞及 NK 細胞的激活因子,属于趋

化因子家族 CC 亚族一个成员。大鼠 MCP-1 cDNA 编码 99 个氨基酸组成的前体蛋白,成熟蛋白只有 76 个氨基酸,是前体蛋白被酶切掉 23 个

氨基酸疏水性信号肽形成的。

MCP-1 被认为在与单核细胞渗透相关的许多疾病有关,包括动脉粥样硬化、类风湿性关节炎和过敏反应等。几例体外动物模型实验表

MCP-1 在炎症反应的过程中扮演着重要的角色。MCP-1 也被证明在包括胃癌、食管上皮磷癌、恶性胶质瘤、卵巢癌、胰腺癌、膀胱癌和

乳腺癌的调控中起作用。

体内分泌 MCP-1 的细胞有:单核细胞、肥大细胞、T 细胞、成骨细胞、成纤维细胞、内皮细胞、表皮细胞、小胶质细胞、星形胶质细

胞及平滑肌细胞等。

检测原理:

本试剂盒采用双抗体夹心ELISA法检测样本中MCP-1的浓度。MCP-1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MCP-1

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大鼠MCP-1抗体后,抗大鼠MCP-1抗体与MCP-1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MCP-1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,MCP-1

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MCP-1 浓度。

大鼠MCP-1定量分析酶联免疫检测试剂盒组成:

组分 规格(96T/48T)

大鼠MCP-1预包被板 12条/6条

标准品稀释液 10ml/5ml

大鼠MCP-1标准品 2/1支(冻干)

大鼠MCP-1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。

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28℃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.如有5X准品稀释液,请按所需量用双蒸水或去离子水稀释(1份加4水)。

4.标准品: 按标签复溶体积加入标准品稀释液复溶使MCP-1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,用标准品稀释液倍比梯度稀释后依次加入检测孔中。(标准曲线取七个点,最高浓度为1000 pg/ml,

标准品稀释液直接加入作为0浓度)

洗涤方法:

自动洗板机或大鼠工洗板: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。不同样本需根据实验

确定稀释倍数,一般血清血浆用标准品稀释液 2 倍稀释。

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20分钟。

10.加入中止液50ul/孔,混匀后即刻测量OD450值。

上海传秋生物科技有限公司 www.chuanqiubio.com大鼠MCP-1参考标准曲线

0

0.5

1

1.5

2

2.5

0 31.25 62.5 125 250 500 1000

大鼠MCP-1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OD1 典型OD2 OD平均值

0 0.092 0.056 0.074

31.25 0.242 0.182 0.212

62.5 0.374 0.322 0.348

125 0.576 0.52 0.548

250 0.911 0.859 0.885

500 1.517 1.469 1.493

1000 2.303 2.159 2.231

大鼠MCP-1参考标准曲线

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8.9pg/ml。

2.特异性:与大鼠的的CCL3/MIP-1α G-CSF、GM-CSF、IFN-γ IL-1α IL-1β 无交叉反应性,与小鼠的MCP-133%的交叉反应性。

3.重复性:板内,板间变异系数均<10%.

参考文献:

1. Frazier-Jessen, M.R. and E.J. Kovacs (1995) J. Immunol. 154:1838.

2. Luo, Y. et al. (1994) J. Immunol. 153:3708.

3. Gu, L. et al. (1997) J. Leukoc. Biol. 62:577.

4. Boring, L. et al. (1996) J. Biol. Chem. 271:7551

5. Bottazzi, B. et al. (1992) J. Immunol. 148:1280.

6.. Volejnikova, S. et al. (1997) Am. J. Pathol. 150:1711.上海传秋生物科技有限公司 www.chuanqiubio.com

ELISA Kit for the Quantitative Analysis of Rat MCP-1

The rat MCP-1 ELISA (enzyme-linked immunosorbent assay) kit is used for detection of rat MCP-1 in cell culture supernatants,rat

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

Monocyte chemotactic protein 1 (MCP-1/CCL2), also known as monocyte chemotactic and activating factor (MCAF), is one of

chemoattractant that was shown to not only monocyte but also T lymphocytes and NK cells, which is a member of the CC subgroup of the

chemokine superfamily . The rat MCP-1 cDNA encodes a 99 amino acid (aa) residue precursor protein with a 23 aa hydrophobic signal

peptide that is cleaved to generate the 76 aa mature protein.

MCP-1 was postulated to play a pathogenic role in a variety of diseases characterized by mononuclear cell infiltration, including

atherosclerosis, rheumatoid arthritis and allergic responses . Several different lines of evidence utilizing in vivo animal models suggest

that MCP-1 is an important player in inflammatory processes. MCP-1 is also upregulated in several cancers including gastric carcinoma,

esophageal squamous cell carcinoma, malignant glioma, and ovarian, pancreatic, bladder, and breast cancers.

MCP-1 produced by several cell types including mononuclear cells, T cells, endothelial cells, fibroblasts, microglia,

osteoblasts,mast cells, bone marrow stromal cells, epithelial cells, astrocytes, and smooth muscle cells.

Principles of the Test

The kits is a solid sandwich enzyme-linked immunosorbent assay for detection of rat MCP-1. An anti-rat MCP-1 monoclonal antibody

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

The rat MCP-1 in specimens or standards would be captured by the coated antibody and the free others were removed by washing. The

rat MCP-1 biotin-conjugated antibody were added and binds to rat MCP-1 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 rat MCP-1 in the original specimen.

Materials provided with the kits:

Reagent 96/48Test Kit

Rat MCP-1 Antibody-Coated Wells 12 strips/6 strips

Standard Diluent 10ml/5ml

Rat MCP-1 Standard 2/1vial(s)

Rat MCP-1 Detetion Antibody 10ml/5ml

Streptavidin-HRP 10ml/5ml

Wash Buffer Concentrate 20× 30ml/15ml

TMB 10ml/5ml

Stop Solution 5ml/3 ml

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 Serumsamples.

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

4. Do not use grossly hemolyzed or lipemic samples.

Precautions for use:

1. Please storage the Kit at 28℃。

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 2528.

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 diluent to the bottle according to the volume of the label and wait 15 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

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 2 fold.

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 TMBLucifugal incubation for 20 minutes at room temperature.

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

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 0.092 0.056 0.074

31.25 0.242 0.182 0.212

62.5 0.374 0.322 0.348

125 0.576 0.52 0.548

250 0.911 0.859 0.885

500 1.517 1.469 1.493

1000 2.303 2.159 2.231

Rat MCP-1 Standard Curve

Sensitivity,Specificity, Repeatability

Sensitivity: repeated assays were evaluated and the minimum detectable dose was8.9 pg/ml.

Specificity : No significant cross-reactivity or interference with rat CCL3/MIP-1α G-CSF、GM-CSF、IFN-γ IL-1α IL-1β and have 33%

cross-reactivity with Mouse MCP-1.

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

REFERENCES:

1. Frazier-Jessen, M.R. and E.J. Kovacs (1995) J. Immunol. 154:1838.

2. Luo, Y. et al. (1994) J. Immunol. 153:3708.

3. Gu, L. et al. (1997) J. Leukoc. Biol. 62:577.

4. Boring, L. et al. (1996) J. Biol. Chem. 271:7551

5. Bottazzi, B. et al. (1992) J. Immunol. 148:1280.

6.. Volejnikova, S. et al. (1997) Am. J. Pathol. 150:1711.

 


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