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人的肽受体酪氨酸激酶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 Mer 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。

人Mer简介：

肽受体酪氨酸激酶 Mer，也被称作 MERTK，是 AXL 酪氨酸受体激酶的三个组成成分中的一个，另外两个成分是 Axl 和 Tyro3。MERTK 是

一个跨膜蛋白，但可由基质金属蛋白酶的裂解，使得膜外部分脱落形成可溶性的形式。

MERTK 一般在单核细胞、上皮来源的细胞及生殖组织中高表达。在一般的 B 淋巴细胞和 T 淋巴细胞中不表达，但在 B 淋巴细胞和 T 淋

巴细胞有肿瘤中有表达。

MERTK通过与Gas6基因的蛋白产物结合而自磷酸化，从而启动下游的信号通路，从而在细胞的增殖、迁移、阻止细胞的调亡等生理过程

中起作用。最近的研究表明，这个AXL家族的酪氨酸激酶受体可能与造血过程、胚胎发育、及睾丸功能的调控、生理平衡、炎症反应，自身

免疫、肿瘤发生及恶化等生理过程有关。

检测原理:

本试剂盒采用双抗体夹心ELISA法检测样本中Mer的浓度。Mer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Mer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Mer抗体后，抗Mer抗体与Mer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Mer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，Mer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Mer浓度。

人Mer定量分析酶联免疫检测试剂盒组成:

组分 规格（96T/48T）

人 Mer 预包被板 12 条/6 条

样本分析缓冲液 5ml/3ml

标准品稀释液 10ml/5ml

人 Mer 标准品 2 支/1 支(冻干粉)

50×Mer 生物素化抗体 210μ l/105μ l

生物素化抗体稀释液 10ml/5ml

HRP 连接的酶结合物 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.标本收集后请分装，若待测样本不能及时检测，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 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.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 Mer 终浓度达到 20ng/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 20ng/ml,标

准品稀释液直接加入作为 0 浓度.）

4.检测抗体，加样前，按1：50配制所需的工作液。

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:

上海传秋生物科技有限公司 www.chuanqiubio.com人Mer参考标准曲线

0

0.5

1

1.5

2

2.5

0 0.625 1.25 2.5 5 10 20

浓度（ng/ml）

O

D值

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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，血清血浆标本请用标准品稀释液从 5～20 倍稀释后加样，用封板胶纸封住，室

温（25-28℃）孵育 120 分钟。

4.洗板 3 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，室温（25-28℃）孵育 60 分钟。

6.洗板 3 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 30 分钟。

8.洗板 3 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.加入终止液 50ul/孔,混匀后即刻测量 OD450 值。

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ng/ml 典型OD值1 典型OD值2 OD平均值

0 0.0522 0.0716 0.0619

0.625 0.2103 0.2373 0.2238

1.25 0.3495 0.4033 0.3764

2.5 0.6248 0.667 0.6459

5 1.0681 1.1087 1.0884

10 1.5362 1.5912 1.5637

20 2.0937 2.1095 2.1016

人Mer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0.45ng/ml。2.特异性：与人的Axl激酶复合物、Dtk激酶复合物以及TrkA激酶复合物无交叉反应性。

3.重复性：板内，板间变异系数均<10%.

参考文献:

1. Shimojima M. et al., 2006, J Virol. 80: 10109-162

2. Hillier LW. et al., 2005, Nature. 434:724-31.

3.. Oppermann FS.et al., 2009, Mol Cell Proteomics 8: 1751-642.

4. Gal A. et al., 2000, Nat Genet. 26: 270-1.

.

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ELISA Kit for the Quantitative Analysis of Human Mer

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

Tyrosine-proteinkinase Mer,also known as MERTK, is a member of the AXL receptor tyrosine kinase subfamily which includes two

other members Axl and Tyro3. MERTK is transmembrane protein which can yield soluble form by proteolytic cleavage of the

extracellular domain via a metalloproteinase.

MERTK is expressed at high levels in monocytes and cells derived from epithelial and reproductive tissues,is not expressed in normal

B lymphocytes or T lymphocytes but is expressed in nuMerous neoplastic B lymphocytes and T lymphocytes.

Binding of Gas6 induces receptor autophosphorylation and downstream signaling pathways that can lead to cell proliferation, migration

or the prevention of apoptosis. Recent studies suggest that this family of tyrosine kinase receptors may be involved in hematopoiesis, e

mbryonic development, regulation of testicular functions. homeostasis, inflammation, and autoimmune responses, as well as

tumorigenesis and malignancy.

Principles of the Test

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

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

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

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

Materials provided with the kits:

reagent 96/48Test Kit

Human Mer Antibody-Coated Wells 12 strips/6 strips

Assay Buffer 5ml/3ml

Standard Diluent 10ml/5ml

Human Mer Standard 2/1vial(s)

50×Mer Detetion Antibody 5ml/2.5ml

Detetion Antibody Diluent 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 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 assay process.

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 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.

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 deionized or distilled water 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 .

4.Prepared detection antibody working solution with detection antibody 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.

上海传秋生物科技有限公司 www.chuanqiubio.comHuman Mer Standard Curve

0

0.5

1

1.5

2

2.5

0 0.625 1.25 2.5 5 10 20

Concentration（ng/ml）

O

p

t

i

c

a

l

D

e

n

s

i

t

y

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3. Add 100ul of standard or sample,Please diluent from 5 to 20 times to serum or plasma. Cover with the Plate Covers provided. Incubate

for 2 hours at room temperature.

4.Three 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. Three 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.Three 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.

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(ng/ml) Typical data 1 Typical data 2 Average

0 0.0522 0.0716 0.0619

0.625 0.2103 0.2373 0.2238

1.25 0.3495 0.4033 0.3764

2.5 0.6248 0.667 0.6459

5 1.0681 1.1087 1.0884

10 1.5362 1.5912 1.5637

20 2.0937 2.1095 2.1016

Human Mer standard curve

Sensitivity, Specificity, Repeatability

Sensitivity: repeated assays were evaluated and the minimum detectable dose was 0.45ng/ml.

Specificity : No significant cross-reactivity or interference with human Axl Kinase complex、Dtk Kinase complex and TrkA Kinase

complex

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

1. Shimojima M. et al., 2006, J Virol. 80: 10109-162

2. Hillier LW. et al., 2005, Nature. 434:724-31.

3.. Oppermann FS.et al., 2009, Mol Cell Proteomics 8: 1751-642.

4. Gal A. et al., 2000, Nat Genet. 26: 270-1.

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