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人的干細(xì)胞因子定量分析酶聯(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(xì)胞培養(yǎng)上清液中的 SCF 濃度。使用前請仔細(xì)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?jiān)谑盏截浺粋€(gè)月之內(nèi)聯(lián)系我們。

SCF 簡介：

干細(xì)胞因子（SCF）是一個(gè)多效性的細(xì)胞因子，還被稱為肥大細(xì)胞生長因子或鋼因子。兩種前體，一種叫做KL-1的前體蛋白分子量為45

kDa，能夠裂解為31 kDa的可溶性蛋白。另一種前體蛋白分子量為32 kDa，也叫KL-2，因?yàn)槿鄙俣鄠€(gè)分裂位點(diǎn)，一般都形成31 kDa的可溶性

蛋白，但也常被裂解為23kDa的效果較差的蛋白。

SCF在性配子的形成、黑色素生成及造血系早期的形成上有重要的作用。無論是體內(nèi)還是體外，SCF都能刺激成熟的肥大細(xì)胞像未成熟

的細(xì)胞分化為成熟的細(xì)胞一樣有效。在原始的造血系細(xì)胞的前體細(xì)胞的培養(yǎng)中，SCF表現(xiàn)出與IL-1、IL-3、IL-6、 IL-7及 Epo有協(xié)同增效

作用，能夠誘導(dǎo)骨髓的紅細(xì)胞系和淋巴細(xì)胞系細(xì)胞的集落形成

檢測原理:

本試劑盒采用雙抗體夾心ELISA法檢測樣本中SCF的濃度。SCF捕獲抗體已預(yù)包被于酶標(biāo)板上，當(dāng)加入標(biāo)本或參考品時(shí)，其中的SCF會(huì)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(dāng)加入生物素化的抗人SCF抗體后，抗人SCF抗體與SCF接合，形成夾心的免疫復(fù)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(biāo)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(huì)

與夾心的免疫復(fù)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SCF將會(huì)形成免疫復(fù)合物，辣根過

氧化物酶會(huì)催化無色的顯色劑氧化成藍(lán)色物質(zhì)，在加入終止液后呈黃色。通過酶標(biāo)儀檢測，讀其450nm處的OD值，SCF濃度與OD450值之間呈

正比，通過參考品繪制標(biāo)準(zhǔn)曲線，對照未知樣本中OD值，即可算出標(biāo)本中SCF濃度。

人SCF定量分析酶聯(lián)免疫檢測試劑盒組成:

組分 規(guī)格（96T/48T）

人SCF預(yù)包被板 12條/6條

樣本分析緩沖液 5ml/3ml

標(biāo)準(zhǔn)品稀釋液 10ml/5ml

人SCF標(biāo)準(zhǔn)品 2支/1支(凍干)

人SCF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/5 ml

中止液 5ml/3 ml

封板膠紙 3/2張

說明書 1份

標(biāo)本收集:

1.標(biāo)本的收集請按下列流程進(jìn)行操作；

A.細(xì)胞上清標(biāo)本離心去除懸浮物后即可；

B.血清標(biāo)本應(yīng)是自然凝固后，取上清，避免在冰箱中凝固血液；

C.血漿標(biāo)本，推薦用EDTA的方法收集若待測樣本不能及時(shí)檢測，

D.標(biāo)本收集后請分裝，凍存于－20℃，避免反復(fù)凍融。

2.血清標(biāo)本不應(yīng)添加任何防腐劑或抗凝劑；

3.標(biāo)本應(yīng)清澈透明，檢測前樣本中如有懸浮物應(yīng)通過離心去除。

4.請勿使用溶血，高血脂或污染的標(biāo)本檢測，否則結(jié)果將不準(zhǔn)確。注：人血清或血漿樣本請用樣本分析緩沖液做倍比稀釋后再檢測。

注意事項(xiàng):

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(biāo)準(zhǔn)品復(fù)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(shí)，請及時(shí)更換槍頭，避免交叉污染。

6.嚴(yán)禁混用不同批號(hào)的試劑盒組份。

7.充分混勻?qū)ΡＷC反應(yīng)結(jié)果的準(zhǔn)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(yīng)，請嚴(yán)格控制在25～28℃。

9.洗滌過程是至關(guān)重要的，洗滌不充分會(huì)使精確度下降并導(dǎo)致結(jié)果誤差較大。

10.試驗(yàn)中標(biāo)準(zhǔn)品和樣本檢測時(shí)建議作雙復(fù)孔。

11.加樣過程中避免氣泡的產(chǎn)生。

12.血清和血漿標(biāo)本的檢測時(shí)，檢測抗體的孵育時(shí)間應(yīng)適當(dāng)延長。

檢測前準(zhǔn)備工作:

1.試劑盒自冰箱中取出后應(yīng)置室溫（25～28℃）平衡20分鐘；每次檢測后剩余試劑請及時(shí)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(zhǔn)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(biāo)準(zhǔn)品: 按標(biāo)簽復(fù)溶體積加入標(biāo)準(zhǔn)品稀釋液復(fù)溶使SCF終濃度達(dá)到2000pg/ml，室溫反應(yīng)，請嚴(yán)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(biāo)準(zhǔn)品稀釋液倍比梯度稀釋后依次加入檢測孔中。（標(biāo)準(zhǔn)曲線取七個(gè)點(diǎn)，最高濃度為2000 pg/ml,

標(biāo)準(zhǔn)品稀釋液直接加入作為0濃度.）

洗滌方法:

自動(dòng)洗板機(jī)或人工洗板：每孔洗滌液為300ul，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(shí)驗(yàn)過程需自備的材料：

1.不同規(guī)格的加樣槍及相應(yīng)的槍頭；

2.酶標(biāo)儀；

3.自動(dòng)洗板機(jī)；

4.去離子水或雙蒸水；

操作步驟:

1.通過計(jì)算并確定一次性實(shí)驗(yàn)所需的板條數(shù)，取出所需板條放置在框架內(nèi)，暫時(shí)用不到板條請放回鋁箔袋密封，保存于4℃。

2.建議設(shè)置本底較正孔，即空白孔,設(shè)置方法為該孔只加TMB顯色液和中止液。每次實(shí)驗(yàn)均需做標(biāo)準(zhǔn)品對照并畫出標(biāo)準(zhǔn)曲線。

3.分別將標(biāo)本或不同濃度標(biāo)準(zhǔn)品(100ul/孔)加入相應(yīng)孔中，用封板膠紙封住反應(yīng)孔，室溫（25～28℃）孵育120分鐘。對于血清或血漿標(biāo)本，

請加入50 ul樣本分析緩沖液后加50 ul標(biāo)本, 如稀釋量大，請將樣本與樣本分析緩沖液等量加入，不足部分用標(biāo)準(zhǔn)品稀釋液補(bǔ)充至100ul。

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(yīng)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(yīng)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SCF 標(biāo)準(zhǔn)曲線

0

1

2

3

0 62.5 125 250 500 1000 2000

人SCF 濃度（pg/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(jié)果判斷:

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

2.每個(gè)標(biāo)準(zhǔn)品或標(biāo)本的OD值應(yīng)減去本底校正孔的OD值。

3.手工繪制標(biāo)準(zhǔn)曲線。以標(biāo)準(zhǔn)品濃度作橫坐標(biāo)，OD值作縱坐標(biāo)，以平滑線連接各標(biāo)準(zhǔn)品的坐標(biāo)點(diǎn)。通過標(biāo)本的OD值可在標(biāo)準(zhǔn)曲線上查出其

濃度。

4.若標(biāo)本OD值高于標(biāo)準(zhǔn)曲線上限，應(yīng)適當(dāng)稀釋后重測，計(jì)算濃度時(shí)應(yīng)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 0.0991 0.1157 0.1074

62.5 0.2915 0.2817 0.2866

125 0.4612 0.536 0.4986

250 0.8753 0.8395 0.8574

500 1.1801 1.1963 1.1882

1000 1.5516 1.53 1.5408

2000 2.0204 2.012 2.0162

人 SCF 參考標(biāo)準(zhǔn)曲線

注意：本圖僅供參考，應(yīng)以同次試驗(yàn)標(biāo)準(zhǔn)品所繪標(biāo)準(zhǔn)曲線計(jì)算標(biāo)本含量。

靈敏度，特異性和重復(fù)性:

1.靈敏度：多次重復(fù)結(jié)果表明，最小檢出量為13.6pg/ml。

2.特異性：與人EGF,Epo,G-CSF,GM-CSF,IL-1α ,IL-1β ,IL-2,IL-4,IL-6,IL-8,LIF,TGF-β 1,TNF-α 等沒有交叉反應(yīng)。

3.重復(fù)性：板內(nèi)，板間變異系數(shù)均<10%.

參考文獻(xiàn):

1. Williams, D.E. et al. (1990) Cell 63:167.

2. Moore, M.A.S. (1991) Blood 78:1.

3. Morrison-Graham, K. and Y. Takahashi (1993) BioEssays 15:77.

4. Temeles, D.S. et al. (1993) Exp. Hematol. 21:388.

5. Ryan, J.J. et al. (1994) J. Neurosci. Res. 37:415.

6. Patchen, M.L. et al. (1994) Exp. Hematol. 22:31.

7. Mitsui, H. et al. (1993) Proc. Natl. Acad. Sci. USA 90:735.

8. Briddell, R.A. et al. (1993) Blood 82:1720.上海傳秋生物科技有限公司 424100.com.cn

ELISA Kit for the Quantitative Analysis of Human SCF

The human SCF ELISA (enzyme-linked immunosorbent assay) kit is used for detection of human SCF in cell culture

supernatants,human serum and plasma.THE ELISA KIT IS FOR RESEARCH USE ONLY

Introduction

stem cell factor (SCF) is pleiotropic cytokine, alternately known as the stem cell factor (SCF), mast cell growth factor (MGF) and

steel-factor (SLF).

A larger 45 kDa form (also called KL-1) and the smaller 32 kDa form (also called KL-2) are cleaved from the precursor to produce

soluble factors. Cleavage of KL-1 gives rise to a 31 kDa soluble form. The splice variant KL-2 lacks the proteolytic cleavage site used to

generate soluble KL-1, but uses a site that is cleaved with lower efficiency to generate a 23 kDa soluble molecule (17, 18).

SCF plays essential roles in gametogenesis, melanogenesis and early stages of hematopoiesis. In vitro and in vivo, SCF can

stimulate the proliferation of mature, as well as the proliferation and maturation of immature, mast cells. On purified primitive human and

mouse hematopoietic precursors, SCF acts in a synergistic manner with various growth factors, such as IL-1, IL-3, IL-6, IL-7, and Epo, to

induce myeloid, erythroid and lymphoid lineage colony formation

Principles of the Test

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

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

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

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

Materials provided with the kits:

reagent 96/48Test Kit

Assay Buffer 5ml/3 ml

Human SCF Antibody-Coated Wells 12 strips/6 strips

Standard Diluent 10 ml/5ml

Human SCF Standard 2/1vial(s)

Human SCF Detetion Antibody 10ml/5ml

Streptavidin-HRP 10ml/5ml

Wash Buffer Concentrate 20× 30ml/15ml

TMB 10ml/5 ml

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 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. 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. 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 assay the serum

sample,you should add 50μ l assay buffer with 50μ l sample into the wells,if the protein concentration is higher than the range of the Kit,

add the same quantitys assay buffer with the sample, the deficiency should be complemented with sample diluent to 100μ l per well.

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.

2.The blank absorbance values of subtract should be deducted.

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

0

1

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0 62.5 125 250 500 1000 2000

Human SCF Concentration(pg/ml)

O

p

i

t

i

c

a

l

D

e

n

s

t

i

y

上海傳秋生物科技有限公司 424100.com.cn

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 0.0991 0.1157 0.1074

62.5 0.2915 0.2817 0.2866

125 0.4612 0.536 0.4986

250 0.8753 0.8395 0.8574

500 1.1801 1.1963 1.1882

1000 1.5516 1.53 1.5408

2000 2.0204 2.012 2.0162

Human SCF standard curve

Sensitivity, Specificity, Repeatability

Sensitivity: repeated assays were evaluated and the minimum detectable dose was 13.6pg/ml.

Specificity: No significant cross-reactivity or interference with human EGF,Epo,G-CSF,GM-CSF,IL-1α ,IL-1β , IL-2, IL-4,

IL-6,IL-8,LIF,TGF-β 1,TNF-α .

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

REFERENCES:

1. Williams, D.E. et al. (1990) Cell 63:167.

2. Moore, M.A.S. (1991) Blood 78:1.

3. Morrison-Graham, K. and Y. Takahashi (1993) BioEssays 15:77.

4. Temeles, D.S. et al. (1993) Exp. Hematol. 21:388.

5. Ryan, J.J. et al. (1994) J. Neurosci. Res. 37:415.

6. Patchen, M.L. et al. (1994) Exp. Hematol. 22:31.

7. Mitsui, H. et al. (1993) Proc. Natl. Acad. Sci. USA 90:735.

8. Briddell, R.A. et al. (1993) Blood 82:1720.