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大鼠白細胞介素 10 定量分析酶聯(lián)免疫檢測試劑盒

本試劑盒僅供科研使用。用于體外定量檢測大鼠血清、血漿或細胞培養(yǎng)上清液中的 IL-1O 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。

IL-1O 簡介：

大鼠白介 10 的 cDNA 編碼 178 個氨基酸的前體蛋白，通過切割掉 18 個信號肽形成成熟的大鼠白介 10 的蛋白，大鼠白介 10（IL－10）

是一個多效性的細胞因子，在抑制激活的 Th1 細胞、NK 細胞和單核/巨噬細胞產(chǎn)生 IL-1, GM-CSF, TNF, IL-6, IL-8, IL-12 和 IFN-γ 等

細胞因子方面起重要的作用。同時在抑制巨噬細胞細胞毒活性性和 B 細胞、肥大細胞和 T 細胞的增殖和分化方面起重要的作用。能產(chǎn)生 IL-10

的細胞很多，包括激活的 Th2 細胞、幼稚的胸腺細胞、單核/巨噬細胞、角化細胞、B 細胞和神經(jīng)膠質(zhì)細胞等。

檢測原理:

本試劑盒采用雙抗體夾心ELISA法檢測樣本中大鼠IL-10的濃度。大鼠IL-10捕獲抗體已預(yù)包被于酶標板上，當加入標本或參考品時，

其中的大鼠IL-10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大鼠IL-10抗體后，抗大鼠IL-10抗體與

大鼠IL-10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(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IL-10將會形成免疫復(fù)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，大鼠IL-10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IL-10濃度。

大鼠IL-10定量分析酶聯(lián)免疫檢測試劑盒組成:

組分 規(guī)格（96T/48T）

大鼠IL-10預(yù)包被板 12條/6條

樣本分析緩沖液 5ml/3ml

標準品稀釋液 10ml/5ml

大鼠IL-10標準品 2支/1支(凍干)

大鼠IL-10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(yīng)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(fù)凍融。

2.血清標本不應(yīng)添加任何防腐劑或抗凝劑；

3.標本應(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，高血脂或污染的標本檢測，否則結(jié)果將不準確。

注：大鼠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:

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(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，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(yīng)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(yīng)，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(dǎo)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(fù)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(yīng)適當延長。

檢測前準備工作:

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(fù)溶體積加入標準品稀釋液復(fù)溶使IL-10終濃度達到4000pg/ml，室溫反應(yīng)，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4000pg/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。

實驗過程需自備的材料：

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(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(yīng)孔中，用封板膠紙封住反應(yīng)孔，室溫（25～28℃）孵育120分鐘。對于血清或血漿標本，

請加入50 ul樣本分析緩沖液后加50 ul標本，如稀釋量大，請將樣本與樣本分析緩沖液等量加入，不足部分用標準品稀釋液補充至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.加入親和素鏈接的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。

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。

上海傳秋生物科技有限公司 424100.com.cn大鼠 IL-10 標準曲線

0

0.5

1

1.5

2

2.5

0 125 250 500 1000 2000 4000

大鼠 IL-10 濃度（pg/ml）

O

D值

上海傳秋生物科技有限公司 424100.com.cn

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.每個標準品或標本的OD值應(yīng)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(yīng)適當稀釋后重測，計算濃度時應(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 0.2531 0.0873 0.1702

125 0.3928 0.3492 0.371

250 0.4129 0.5117 0.4623

500 0.8004 0.7544 0.7774

1000 1.1251 1.0853 1.1052

2000 1.4623 1.5241 1.4932

4000 2.1302 2.1088 2.1195

大鼠IL-1O參考標準曲線

注意：本圖僅供參考，應(yīng)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(fù)性:

1.靈敏度：多次重復(fù)結(jié)果表明，最小檢出量為28pg/ml。

2.特異性：與GDNF,GM-CSF,IFN-γ ,IL-1α ,IL-1β ,IL-2 ,IL-4,IL-6,PDGF-BB,TNF-α ,human IL-10, mouse IL-10

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:

1. Fiorentino, D.F. et al. (1989) J. Exp. Med. 170:2081.

2. Marconi, M. et al. (1998) Clin. Exp. Immunol. 112:528.

3. Olikowsky, T. et al. (1997) Immunology 91:104.

4. Panuska, J.R. et al. (1995) J. Clin. Invest. 96:2445.

5. Sato, T. et al. (1996) Clin. Cancer Res. 2:1383.

6. Capsoni, F. et al. (1998) Cell. Immunol. 189:51.

7. Liu, Y. et al. (1994) J. Immunol. 152:1821.上海傳秋生物科技有限公司 424100.com.cn

ELISA Kit for the Quantitative Analysis of Rat IL-10

The rat IL-10 ELISA (enzyme-linked immunosorbent assay) kit is used for detection of rat IL-10 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

Rat IL-10 cDNA encodes a 178 amino acid residue precursor protein that contains an 18 amino acid signal sequence and a 160

amino acid mature protein. Rat interleukin-10 is a pleiotropic cytokine playing an important role by activated Th1 cells, NK cells, and

monocytes/macrophages which be inhibited the production of a number of cytokines (including IL-1, GM-CSF, TNF, IL-6, IL-8, IL-12 and

IFN- IL-10 has also been shown to inhibit macrophage cytotoxic activity and to stimulate the proliferation and differentiation of B cells,

mast cells, and thymic T cells. Rat IL-10 is produced by a wide variety of cells, including activated Th2 cells, fetal thymocytes,

monocytes/macrophages, keratinocytes, B cells, and glial cells.

Principles of the Test

The kits is a solid sandwich enzyme-linked immunosorbent assay for detection of rat IL-10. An anti-rat IL-10 monoclonal antibody has

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

rat IL-10 in specimens or standards would be captured by the coated antibody and the free others were removed by washing. The rat

IL-10 biotin-conjugated antibody were added and binds to rat IL-10 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 IL-10 in the original specimen.

Materials provided with the kits:

reagent 96/48Test Kit

Rat IL-10 Antibody-Coated Wells 12 strips/6 strips

Assay Buffer 5ml/3ml

Standard Diluent 10ml/5ml

Rat IL-10 Standard 8/4vial(s)

Rat IL-10 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. 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 into 300ul 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.

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 0.2531 0.0873 0.1702

125 0.3928 0.3492 0.371

250 0.4129 0.5117 0.4623

500 0.8004 0.7544 0.7774

1000 1.1251 1.0853 1.1052

2000 1.4623 1.5241 1.4932

4000 2.1302 2.1088 2.1195

Rat IL-1O standard curve

Sensitivity, Specificity, Repeatability

Sensitivity: repeated assays were evaluated and the minimum detectable dose was 28pg/ml.

Specificity: No significant cross-reactivity or interference with Mouse GDNF,GM-CSF,IFN-γ ,IL-1α ,IL-1

β ,IL-2 ,IL-4,IL-6,PDGF-BB,TNF-α ,human IL-10, mouse IL-10

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

REFERENCES:

1. Fiorentino, D.F. et al. (1989) J. Exp. Med. 170:2081.

2. Marconi, M. et al. (1998) Clin. Exp. Immunol. 112:528.

3. Olikowsky, T. et al. (1997) Immunology 91:104.

4. Panuska, J.R. et al. (1995) J. Clin. Invest. 96:2445.

5. Sato, T. et al. (1996) Clin. Cancer Res. 2:1383.

6. Capsoni, F. et al. (1998) Cell. Immunol. 189:51.

7. Liu, Y. et al. (1994) J. Immunol. 152:1821.