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人巨噬細胞刺激蛋白定量分析酶聯(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MSP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。

MSP簡介：

人巨噬細胞刺激蛋白(MSP)，也叫肝素樣蛋白，屬于肝素生長因子家族的蛋白之一。巨噬細胞刺激蛋白由肝實質(zhì)細胞產(chǎn)生前體蛋白，

前體蛋白被裂解后產(chǎn)生45~62氨基酸的α 亞基和25~35氨基酸的β 亞基通過二硫鏈形成異二聚體活性形式。

巨噬細胞刺激蛋白的功能是對巨噬細胞介入炎癥反應有正負兩方面的作用。巨噬細胞刺激蛋白能刺激角蛋白的增殖，腎小管內(nèi)皮細胞

增殖，腎小球膜系細胞的增殖以及破骨細胞在骨再吸收過程中的功能。巨噬細胞刺激蛋白能夠抑制造血系統(tǒng)中髓系原始造血細胞的增殖和

分化，但能增強巨核細胞和類紅細胞的分化。巨噬細胞刺激蛋白作為一個神經(jīng)因子，能夠維持神經(jīng)細胞的存活和促進感覺神經(jīng)元和交感神

經(jīng)元的神經(jīng)突的延伸。

在急性腎壞死、支氣管擴張和肉狀瘤病引起的細小支氣管病變中，巨噬細胞刺激蛋白在血清中的深度會有顯著提高。

檢測原理:

本試劑盒采用雙抗體夾心ELISA法檢測樣本中MSP的濃度。MSP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MSP 會

與捕獲抗體結合，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MSP 抗體后，抗人MSP 抗體與MSP 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MSP 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，MSP 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MSP 濃度。

人 MSP 定量分析酶聯(lián)免疫檢測試劑盒組成:

組分 規(guī)格（96T/48T）

人MSP預包被板 12條/6條

5X標準品稀釋液 10ml/5ml

人MSP標準品 4/2 支(凍干)

人MSP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，否則結果將不準確。

注：人血清或血漿樣本請用標準品稀釋液做稀釋后再檢測。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.充分混勻對保證反應結果的準性很重要，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.洗滌過程是至關重要的，洗滌不充分會使精確度下降并導致結果誤差較大。

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. 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: 按標簽復溶體積加入 1X 標準品稀釋液復溶使 MSP 終濃度達到 10ng/ml，室溫反應，請嚴格控制在 25～28℃，靜置 15~20 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 10ng/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。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80~800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。

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。

上海傳秋生物科技有限公司 424100.com.cn人MSP參考標準曲線

0

0.5

1

1.5

2

2.5

0 0.3125 0.625 1.25 2.5 5 10

人MSP濃度（ng/ml)

O

D值

上海傳秋生物科技有限公司 424100.com.cn

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。

結果判斷:

1.復孔的值在20%的差異范圍內(nèi)結果才有效，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ng/ml 典型OD值1 典型OD值2 OD平均值

0 0.1421 0.1157 0.1289

0.3125 0.2963 0.2791 0.2877

0.625 0.4429 0.3929 0.4179

1.25 0.6433 0.6467 0.645

2.5 0.9927 0.9639 0.9783

5 1.4942 1.4436 1.4689

10 2.1006 2.0542 2.0774

人MSP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：多次重復結果表明，最小檢出量為96pg/ml。

2.特異性：與人的HGF、MSP-a、MSP-β 及小鼠MSP無交叉反應性。

3.重復性：板內(nèi)，板間變異系數(shù)均<10%.

參考文獻:

1. Han, S. et al. (1991) Biochemistry 30:9768.

2. Liu, Q.P. et al. (1999) J. Immunol. 163:6606.

3. Muraoka, R.S. et al. (1999) Endocrinology 140:187.

4. Danilkovitch, A. et al. (1999) J. Biol. Chem. 274:29937.

5. Kurihara, N. et al. (1998) Exp. Hematol. 26:1080.

6. Bhatt, A.S. et al. (2007) Proc. Natl. Acad. Sci. 104:5771.上海傳秋生物科技有限公司 424100.com.cn

ELISA Kit for the Quantitative Analysis of Human MSP

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

Macrophage stimulating protein (MSP), also known as HGF-like protein, is a member of the HGF family of growth factors (1, 2). MSP

is secreted as an inactive pro-protein by hepatocytes (4, 5). The pro-MSP can be cleaved and yield biologically active disulfide linked

heterodimers consisting of a 45-62 kDa alpha chain and a 25-35 kDa beta chain (6, 7).

The function of MSP is both positive and negative effects on macrophage mediated inflammation (1, 2). MSP promotes the

proliferation of keratinocytes, renal tubule epithelial cells, and renal mesangial cells (14, 30, 31), and enhances osteoclast mediated bone

resorption (32). MSP inhibits the differentiation and proliferation of early hematopoietic myeloid progenitors (33) but enhances the

differentiation of megakaryocytes and erythroid cells (34, 35). MSP functions as a neurotrophic factor, promoting neuronal survival as

well as neurite extension and branching of sensory and sympathetic neurons (36, 37).

MSP levels are elevated in the serum during acute renal failure (14) as well as in the bronchiolar lavage fluid of sarcoidosis and

bronchiectasis patients (15, 16).

Principles of the Test

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

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

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

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

Materials provided with the kits:

reagent 96/48Test Kit

Human MSP Antibody-Coated Wells 12 strips/6 strips

Standard Diluent 10ml/5ml

Human MSP Standard 4/2vial(s)

Human MSP 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 in the range of 80 ~ 800 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.

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

0

0.5

1

1.5

2

2.5

0 0.3125 0.625 1.25 2.5 5 10

Human MSP Concentration（ng/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

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

0 0.1421 0.1157 0.1289

0.3125 0.2963 0.2791 0.2877

0.625 0.4429 0.3929 0.4179

1.25 0.6433 0.6467 0.645

2.5 0.9927 0.9639 0.9783

5 1.4942 1.4436 1.4689

10 2.1006 2.0542 2.0774

Human MSP standard curve

Sensitivity,Specificity, Repeatability

Sensitivity: repeated assays were evaluated and the minimum detectable dose was 96pg/ml.

Specificity : No significant cross-reactivity or interference with human HGF、MSP-a、MSP-β and Mouse MSP

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

REFERENCES:

1. Han, S. et al. (1991) Biochemistry 30:9768.

2. Liu, Q.P. et al. (1999) J. Immunol. 163:6606.

3. Muraoka, R.S. et al. (1999) Endocrinology 140:187.

4. Danilkovitch, A. et al. (1999) J. Biol. Chem. 274:29937.

5. Kurihara, N. et al. (1998) Exp. Hematol. 26:1080.

6. Bhatt, A.S. et al. (2007) Proc. Natl. Acad. Sci. 104:5771.