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人白細胞活化黏附因子定量分析酶聯(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ALCAM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。

人ALCAM簡介：

白細胞活化黏附因子(ALCAM)也叫CD166，屬于免疫球蛋白超家族且是 I 型膜糖蛋白。白細胞活化黏附因子有570個氨基酸構(gòu)成5個細胞

間免疫球蛋白樣結(jié)構(gòu)域，這些結(jié)構(gòu)域能與自身或其它蛋白（CD6）結(jié)合而使細胞相互作用。但在內(nèi)皮細胞內(nèi)的分泌型的蛋白只有437個蛋白構(gòu)

成。

白細胞活化黏附因子可由多種組織和細胞產(chǎn)生，這些細胞包括：胸腺內(nèi)皮細胞、血管內(nèi)皮細胞、活化的淋巴細胞和單核細胞，甚至單核

細胞產(chǎn)生的樹突狀細胞。

白細胞活化黏附因子能通過與自身及CD6、NgCAM、甚至某種腦蛋白的相互結(jié)合而起作用。與某種腦蛋白的的結(jié)合使其在突軸的延伸過程

中起作用。白細胞活化黏附因子(ALCAM)與CD6的結(jié)合使其在T細胞的發(fā)育，調(diào)控方面扮演重要的作用。當然，白細胞活化黏附因子(ALCAM)也

可與T細胞和B細胞結(jié)合而活化白細胞。最近研究表明，白細胞活化黏附因子(ALCAM)可使黑色素瘤的轉(zhuǎn)移顯著升高，提示白細胞活化黏附因

子(ALCAM) 可能參與腫瘤轉(zhuǎn)移過程。

檢測原理:

本試劑盒采用雙抗體夾心ELISA法檢測樣本中ALCAM的濃度。ALCAM捕獲抗體已預包被于酶標板上，當加入標本或參考品時，其中的ALCAM

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。當加入生物素化的抗人ALCAM 抗體后，抗人ALCAM 抗體與ALCAM 接合，形成

夾心的免疫復合物，其它游離的成分通過洗滌的過程被除去。隨后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(jié)合，親合

素連接的酶就會與夾心的免疫復合物連接起來；其它游離的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ALCAM 將會形成

免疫復合物，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。通過酶標儀檢測，讀其450nm處的OD值，ALCAM

濃度與OD450值之間呈正比，通過參考品繪制標準曲線，對照未知樣本中OD值，即可算出標本中ALCAM 濃度。

人 ALCAM 定量分析酶聯(lián)免疫檢測試劑盒組成:

組分 規(guī)格（96T/48T）

人ALCAM預包被板 12條/6條

5╳標準品稀釋液 10ml/5ml

人ALCAM標準品 4/2支(凍干)

人ALCAM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后即可；

B.血清標本應是自然凝固后，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，標本收集后請分裝，凍存于－20℃，避免反復凍融。

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，高血脂或污染的標本檢測，否則結(jié)果將不準確。

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.標準品復溶加樣后，剩余部份請丟棄。

4.底物請勿接觸氧化劑和金屬。

5.加樣時，請及時更換槍頭，避免交叉污染。

6.嚴禁混用不同批號的試劑盒組份。

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

8.室溫反應，請嚴格控制在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致結(jié)果誤差較大。

10.試驗中標準品和樣本檢測時建議作雙復孔。

11.加樣過程中避免氣泡的產(chǎn)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后應置室溫（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: 按標簽復溶體積加入1X標準品稀釋液復溶使ALCAM終濃度達到4000pg/ml，室溫反應，請嚴格控制在25～28℃，靜置10~15分鐘后

輕輕混懸（建議抽吸幾次）待徹底溶解，用標準品稀釋液倍比梯度稀釋后依次加入檢測孔中。（標準曲線取七個點，最高濃度為4000 pg/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300μ l，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.建議設置本底較正孔，即空白孔,設置方法為該孔只加TMB顯色液和中止液。每次實驗均需做標準品對照并畫出標準曲線。

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60~600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分鐘。

上海傳秋生物科技有限公司 424100.com.cn人ALCAM參考標準曲線

0

0.5

1

1.5

2

2.5

0 125 250 500 1000 2000 4000

人ALCAM濃度(pg/ml)

O

D值

上海傳秋生物科技有限公司 424100.com.cn

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.加入終止液50ul/孔,混勻后即刻測量OD450值。

結(jié)果判斷:

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

2.每個標準品或標本的OD值應減去本底校正孔的OD值。

3.手工繪制標準曲線。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值高于標準曲線上限，應適當稀釋后重測，計算濃度時應乘以稀釋倍數(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 0.0931 0.0677 0.0804

125 0.2759 0.2427 0.2593

250 0.3989 0.3653 0.3821

500 0.7013 0.6435 0.6724

1000 1.0428 0.986 1.0144

2000 1.5119 1.4383 1.4751

4000 2.0878 2.0286 2.0582

人ALCAM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，特異性和重復性:

1.靈敏度：多次重復結(jié)果表明，最小檢出量為37pg/ml。

2.特異性：與人E-Selectin、ICAM-2、ICAM-3、P-Selectin及小鼠的 ALCAM沒有交叉反應。

3.重復性：板內(nèi)，板間變異系數(shù)均<10%.

參考文獻:

1. Cayrol, R. et al. (2008) Nat. Immunol. 9:137.

2. Degen, W.G. et al. (1998) Am. J. Pathol. 152:805.

3．Van, K. L. et al., 2001, J. Biol. Chem. 276: 25783 - 257890.

4．Hirata, H. et al., 2006, Cells Tissues Organs. 184 (3-4):172-180.

5. Buhusi, M. et al. (2009) J. Neurosci. 29:15630.上海傳秋生物科技有限公司 424100.com.cn

ELISA Kit for the Quantitative Analysis of Human ALCAM

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

ALCAM (activated leukocyte cell adhesion molecule) also known as CD166 is a type I membrane glycoprotein and a member of the

immunoglobulin superfamily. ALCAM / CD166 has 570 aa With five extracellular immunoglobulin-like domains which facilitates

heterophilic (ALCAM-CD6) and homophilic (ALCAM-ALCAM) cell-cell interactions. while the secreted form is 437aa in endothelial cells

ALCAM / CD166 expressed in a wide variety of cells includeing thymic epithelium, microvascular endothelium, activated lymphocytes

and monocytes, and monocyte-derived dendritic cells

ALCAM can bind itself homo typically and is also capable of binding CD6, NgCAM, and other unidentified brain proteins, and thus is

Involved in neurite extension by neurons via heterophilic and homophilic manners. ALCAM / CD6 interaction plays a role in T cell

development and T cell regulation, as well as in the binding of T- cells and B-cells to activated leukocytes. Further, ALCAM has also been

observed to be upregulated on highly metastasizing melanoma cell lines, suggesting a role in tumor migration.

Principles of the Test

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

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

pipetted into wells. The human ALCAM in specimens or standards would be captured by the coated antibody and the free others were

removed by washing. The human ALCAM biotin-conjugated antibody were added and binds to human ALCAM 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 ALCAM in the

original specimen.

Materials provided with the kits:

reagent 96/48Test Kit

Human ALCAM Antibody-Coated Wells 12 strips/6 strips

Standard Diluent 10ml/5ml

Human ALCAM Standard 4/2vial(s)

Human ALCAM 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.

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 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 wait15 minutes for complete dissolution.

Incubation temperature should be 25- 28℃。.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 if is plasma serum

samples, different sample dilution ratio is different, generally range in 60 ~ 600 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 optical 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.

上海傳秋生物科技有限公司 424100.com.cnHuman ALCAM Standard Curve

0

0.5

1

1.5

2

2.5

0 125 250 500 1000 2000 4000

Human ALCAM Concentration(pg/ml)

O

p

t

i

c

a

l

D

e

n

s

i

t

y

上海傳秋生物科技有限公司 424100.com.cn

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 0.0931 0.0677 0.0804

125 0.2759 0.2427 0.2593

250 0.3989 0.3653 0.3821

500 0.7013 0.6435 0.6724

1000 1.0428 0.986 1.0144

2000 1.5119 1.4383 1.4751

4000 2.0878 2.0286 2.0582

Human ALCAM Standard Curve

Sensitivity,Specificity, Repeatability

Sensitivity: repeated assays were evaluated and the minimum detectable dose was 37pg/ml.

Specificity : No significant cross-reactivity or interference with human E-Selectin、ICAM-2、ICAM-3、P-Selectin and Mouse ALCAM.

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

REFERENCES:

1. Cayrol, R. et al. (2008) Nat. Immunol. 9:137.

2. Degen, W.G. et al. (1998) Am. J. Pathol. 152:805.

3．Van, K. L. et al., 2001, J. Biol. Chem. 276: 25783 - 257890.

4．Hirata, H. et al., 2006, Cells Tissues Organs. 184 (3-4):172-180.

5. Buhusi, M. et al. (2009) J. Neurosci. 29:15630.