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人自然殺傷細胞刺激因子定量分析酶聯免疫檢測試劑盒

本試劑盒僅供科研使用 。用於體外定量檢測人血清 、血漿或細胞培養上清液中的 IL-12P70 濃度 。使用前請仔細閱讀說明書並檢查試劑

組分是否完整 。如有產品包裝破損或質量投拆 ，請在收到貨一個月之內聯係尊龍凱時 。

IL-12P70簡介 ：

IL-12P70 也稱為自然殺傷細胞刺激因子（NKSF）或細胞毒性淋巴細胞成熟因子(CLMF)主要是由受激發的巨噬細胞產生的多效性細胞因

子 。IL-12P70 還可由樹突狀細胞 、單核細胞、郎格罕細胞 、嗜中性粒細胞和角化細胞等產生 。

具生物學活性的人 IL-12P70 是二硫鍵連接一個 40 kDa (p40)和 35 kDa (p35)的亞基的異型複合體 ，即 70 kDa (p70) 。成熟的人 40 kDa

(p40)亞基有 13 個半胱氨酸和 5 個可糖基化的 N 末端的 N313 個氨基酸的蛋白 。無論是 p40 還是 p35 亞基都不具有 IL－12 的生物學活性 ，

但由兩個 p40 亞基構成的同型複合物可與 IL-12P70 受體結合 ，從而充當 IL-12P70 的拮抗劑 。雖然小鼠的 IL-12P70 對人和小鼠細胞都有活

性 ，但人的 IL-12P70 隻對人細胞有生物活性 。

IL-12P70對T淋巴細胞和自然殺傷(NK)細胞有多種作用 ，這些作用包括在T細胞和NK細胞中誘導幹擾素和腫瘤壞死因子的生產 ，提高T細

胞和NK細胞毒素的活性的以及刺激T細胞和NK細胞的分化 。IL-12P70是IFN-γ 的強烈誘導物 ，但誘導出的IFN-γ 反過刺激吞噬細胞產生

IL-12P70和其它促炎性因子 ，這樣 ，由IL-12P70誘導的IFN-γ 就在感染時促炎症反應中反饋放大的機理中扮演重要的角色 。IL-12P70 在通

過提高Th1細胞介導的免疫反應中有重要作用 。

檢測原理:

本試劑盒采用雙抗體夾心ELISA法檢測樣本中IL-12P70的濃度 。IL-12P70捕獲抗體已預包被於酶標板上 ，當加入標本或參考品時 ，其中

的IL-12P70 會與捕獲抗體結合 ，其它遊離的成分通過洗滌的過程被除去 。當加入生物素化的抗人IL-12P70 抗體後 ，抗人IL-12P70 抗體與

IL-12P70 接合 ，形成夾心的免疫複合物 ，其它遊離的成分通過洗滌的過程被除去 。隨後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複合物連接起來 ；其它遊離的成分通過洗滌的過程被除去 。最後加入顯色劑 ，若樣本中

存在IL-12P70 將會形成免疫複合物 ，辣根過氧化物酶會催化無色的顯色劑氧化成藍色物質 ，在加入終止液後呈黃色 。通過酶標儀檢測 ，讀

其450nm處的OD值 ，IL-12P70 濃度與OD450值之間呈正比 ，通過參考品繪製標準曲線 ，對照未知樣本中OD值 ，即可算出標本中IL-12P70 濃度 。

人 IL-12P70 定量分析酶聯免疫檢測試劑盒組成:

組分 規格（96T/48T）

人IL-12P70預包被板 12條/6條

樣本分析緩衝液 5ml/3ml

標準品稀釋液 10ml/5ml

人IL-12P70標準品 2/1支(凍幹)

人IL-12P70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後即可 ；

B.血清標本應是自然凝固後 ，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 ，標本收集後請分裝 ，凍存於－20℃ ，避免反複凍融 。

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 ，高血脂或汙染的標本檢測 ，否則結果將不準確 。

注 ：人血清或血漿樣本請用樣本分析緩衝液做倍比稀釋後再檢測 。

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後再配製工作液 。

3.標準品複溶加樣後 ，剩餘部份請丟棄 。

4.底物請勿接觸氧化劑和金屬 。

5.加樣時 ，請及時更換槍頭 ，避免交叉汙染 。

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 ，在加液後請輕輕叩擊邊緣以保證混勻 。

8.室溫反應 ，請嚴格控製在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並導致結果誤差較大 。

10.試驗中標準品和樣本檢測時建議作雙複孔 。

11.加樣過程中避免氣泡的產生 。

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後應置室溫（25～28℃）平衡20分鍾 ；每次檢測後剩餘試劑請及時於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標準品稀釋液 ，請按所需量用雙蒸水或去離子水稀釋備用 。

4.標準品: 按標簽複溶體積加入標準品稀釋液複溶使 IL-12P70 終濃度達到 1000pg/ml ，室溫反應 ，請嚴格控製在 25～28℃ ，靜置 10~15 分

鍾後輕輕混懸（建議抽吸幾次）待徹底溶解 ，用標準品稀釋液倍比梯度稀釋後依次加入檢測孔中 。（標準曲線取七個點 ，最高濃度為 1000

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 。最後一次洗板完成後將板倒扣著在厚吸水紙上用力拍

幹 。

實驗過程需自備的材料 ：

1.不同規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.通過計算並確定一次性實驗所需的板條數 ，取出所需板條放置在框架內 ，暫時用不到板條請放回鋁箔袋密封 ，保存於4℃ 。

2.建議設置本底較正孔 ，即空白孔,設置方法為該孔隻加TMB顯色液和中止液 。每次實驗均需做標準品對照並畫出標準曲線 。

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分鍾 。對於血清或血漿標本 ，

請加入50 ul樣本分析緩衝液後加50 ul標本 ，如稀釋量大 ，請將樣本與樣本分析緩衝液等量加入 ，不足部分用標準品稀釋液補充至100ul 。

上海尊龍凱時生物科技有限公司 www.ruianjituan.com人I L-1 2P7 0參考標準曲線

0

0.5

1

1.5

2

2.5

0 31.25 62.5 125 250 500 1000

濃度（p g/m l）

O

D值

上海尊龍凱時生物科技有限公司 www.ruianjituan.com

4.洗板5次 ，且最後一次置厚吸水紙上拍幹 。

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分鍾 。

6.洗板5次 ，且最後一次置厚吸水紙上拍幹 。

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分鍾 。

8.洗板5次 ，且最後一次置厚吸水紙上拍幹 。

9.加入顯色劑TMB100ul/孔 ，避光室溫（25～28℃）孵育20分鍾 。

10.加入終止液50ul/孔,混勻後即刻測量OD450值 。

結果判斷:

1.複孔的值在20%的差異範圍內結果才有效 ，複孔的值平均後可作為測量值 。

2.每個標準品或標本的OD值應減去本底校正孔的OD值 。

3.手工繪製標準曲線 。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值高於標準曲線上限 ，應適當稀釋後重測 ，計算濃度時應乘以稀釋倍數 。

典型數值和參考曲線

濃度pg/ml 典型OD值1 典型OD值2 OD平均值

0 0.0866 0.0682 0.0774

31.25 0.2046 0.1562 0.1804

62.5 0.3617 0.3203 0.341

125 0.6452 0.5734 0.6093

250 1.0872 0.9636 1.0254

500 1.59 1.5018 1.5459

1000 2.1676 2.0466 2.1071

人IL-12P70參考標準曲線

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 ，特異性和重複性:

1.靈敏度 ：多次重複結果表明 ，最小檢出量為5.8pg/ml 。

2.特異性 ：與人的G-CSF 、IL-6 、IL-12/IL-23 p40及小鼠IL-12 p70 、IL-12/IL-23 p40無交叉反應性。

3.重複性 ：板內 ，板間變異係數均<10%.

參考文獻:

1. Verma, N.D. et al. (2004) SwissProt Accession #:Q9R103.2. Hamza, T. et al. (2010) Int. J. Mol. Sci. 11:789.

3. Shortman, K. and W. Heath (2010) Immunol. Rev. 234:18.

4. Gearing, D.P. and D. Cosman (1991) Cell 66:9.

5. Khalife, J. et al. (1998) Eur. Cytokine Netw. 9:69.

6. Kang, K. et al. (1996) J. Immunol. 156:1402.

ELISA Kit for the Quantitative Analysis of Human IL-12P70

上海尊龍凱時生物科技有限公司 www.ruianjituan.com上海尊龍凱時生物科技有限公司 www.ruianjituan.com

The human IL-12P70 ELISA (enzyme-linked immunosorbent assay) kit is used for detection of human IL-12P70 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

IL-12P70 pleiotropic cytokine, formerly termed cytotoxic lymphocyte maturation factor (CLMF) or natural killer cell stimulatory factor

(NKSF), which is produced primarily by stimulated macrophages. IL-12P70 may be produced by cells include dendritic cells, monocytes ,

Langerhans cells, neutrophils, and keratinocytes.Biologically.

Active human IL-12P70 is a disulfide-linked, 70 kDa (p70) heterodimeric glycoprotein composed of a 40 kDa (p40) subunit and a 35

kDa (p35) subunit. Mature human p40 subunit has 313 aa, with 13 cysteines and five potential N-linked glycosylation sites. Both p40 and

p35 subunits have not activity same with IL-12P70, While the p40 homodimer has been shown to bind the IL-12P70 receptor and is an

IL-12P70 antagonist .Although mouse IL-12P70 is active on both human and mouse cells, human IL-12P70 is only active on human cells.

IL-12P70 exerts a variety of biological effects on T and natural killer cells. Some of these effects include the induction of IFN-γ and TNF-α

production by T and NK cells, the enhancement of cytotoxic activity of T and NK cells and the stimulation of T and NK cell proliferation. As

a potent inducer of interferon gamma (IFNγ) production, the IFNγ then enhances the ability of the phagocytic cells to produce IL-12P70

and other proinflammatory cytokines. Thus, IL-12P70 induced IFNγ acts in a positive feedback loop that represents an important

amplifying mechanism in the inflammatory response to infections. IL-12P70 has also been shown to be a central mediator of the

cell-mediated immune response by promoting Th1 development .

Principles of the Test

The kits is a solid sandwich enzyme-linked immunosorbent assay for detection of human IL-12P70. An anti-human IL-12P70

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

pipetted into wells. The human IL-12P70 in specimens or standards would be captured by the coated antibody and the free others were

removed by washing. The human IL-12P70 biotin-conjugated antibody were added and binds to human IL-12P70 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 IL-12P70 in the

original specimen.

Materials provided with the kits:

reagent 96/48Test Kit

Assay Buffer 5ml/3 ml

Human IL-12P70 Antibody-Coated Wells 12 strips/6 strips

Standard Diluent 10ml/5ml

Human IL-12P70 Standard 2/1vial(s)

Human IL-12P70 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 remnant 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 demonized water.

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 temperatureIf 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 diluents 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 0.0866 0.0682 0.0774

31.25 0.2046 0.1562 0.1804

62.5 0.3617 0.3203 0.341

125 0.6452 0.5734 0.6093

250 1.0872 0.9636 1.0254

500 1.59 1.5018 1.5459

1000 2.1676 2.0466 2.1071

Human IL-12P70 standard curve

Sensitivity,Specificity, Repeatability

Sensitivity: repeated assays were evalsuated and the minimum detectable dose was 5.8pg/ml.

Specificity : No significant cross-reactivity or interference with human G-CSF 、IL-6 、IL-12/IL-23 p40 and Mouse IL-12 p70 、IL-12/IL-23

p40

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

REFERENCES:

1. Verma, N.D. et al. (2004) SwissProt Accession #:Q9R103.

2. Hamza, T. et al. (2010) Int. J. Mol. Sci. 11:789.

3. Shortman, K. and W. Heath (2010) Immunol. Rev. 234:18.

4. Gearing, D.P. and D. Cosman (1991) Cell 66:9.

5. Khalife, J. et al. (1998) Eur. Cytokine Netw. 9:69.

6. Kang, K. et al. (1996) J. Immunol. 156:1402.