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人自然杀伤细胞刺激因子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 IL-12P70 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尊龙凯时人生就是搏。
IL-12P70简介：
IL-12P70 也称为自然杀伤细胞刺激因子（NKSF）或细胞毒性淋巴细胞成熟因子(CLMF)主要是由受激发的巨噬细胞产生的多效性细胞因
子。IL-12P70 还可由树突状细胞 、单核细胞、郎格罕细胞 、嗜中性粒细胞和角化细胞等产生。
具生物学活性的人 IL-12P70 是二硫键连接一个 40 kDa (p40)和 35 kDa (p35)的亚基的异型复合体 ，即 70 kDa (p70) 。成熟的人 40 kDa
(p40)亚基有 13 个半胱氨酸和 5 个可糖基化的 N 末端的 N313 个氨基酸的蛋白 。无论是 p40 还是 p35 亚基都不具有 IL－12 的生物学活性 ，
但由两个 p40 亚基构成的同型复合物可与 IL-12P70 受体结合 ，从而充当 IL-12P70 的拮抗剂 。虽然小鼠的 IL-12P70 对人和小鼠细胞都有活
性 ，但人的 IL-12P70 只对人细胞有生物活性。
IL-12P70对T淋巴细胞和自然杀伤(NK)细胞有多种作用 ，这些作用包括在T细胞和NK细胞中诱导干扰素和肿瘤坏死因子的生产 ，提高T细
胞和NK细胞毒素的活性的以及刺激T细胞和NK细胞的分化。IL-12P70是IFN-γ 的强烈诱导物，但诱导出的IFN-γ 反过刺激吞噬细胞产生
IL-12P70和其它促炎性因子 ，这样 ，由IL-12P70诱导的IFN-γ 就在感染时促炎症反应中反馈放大的机理中扮演重要的角色 。IL-12P70 在通
过提高Th1细胞介导的免疫反应中有重要作用 。
检测原理:
本试剂盒采用双抗体夹心ELISA法检测样本中IL-12P70的浓度。IL-12P70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IL-12P70 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人IL-12P70 抗体后，抗人IL-12P70 抗体与
IL-12P70 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IL-12P70 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，IL-12P70 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IL-12P70 浓度。
人 IL-12P70 定量分析酶联免疫检测试剂盒组成:
组分 规格（96T/48T）
人IL-12P70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IL-12P70标准品 2/1支(冻干)
人IL-12P70生物素化抗体 10ml/5ml
亲和素连接的HRP酶 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.若待测样本不能及时检测 ，标本收集后请分装 ，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 ：人血清或血浆样本请用样本分析缓冲液做倍比稀释后再检测。
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标准品稀释液，请按所需量用双蒸水或去离子水稀释备用 。
4.标准品: 按标签复溶体积加入标准品稀释液复溶使 IL-12P70 终浓度达到 1000pg/ml ，室温反应 ，请严格控制在 25～28℃ ，静置 10~15 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 1000
pg/ml,标准品稀释液直接加入作为 0 浓度.）
洗涤方法:
自动洗板机或人工洗板 ：每孔洗涤液为300μ l ，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:
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 ，用封板胶纸封住反应孔 ，室温（25～28℃）孵育120分钟。对于血清或血浆标本 ，
请加入50 ul样本分析缓冲液后加50 ul标本，如稀释量大 ，请将样本与样本分析缓冲液等量加入 ，不足部分用标准品稀释液补充至100ul。
上海尊龙凯时人生就是搏生物科技有限公司 www.hualeizs.com人I L-1 2P7 0参考标准曲线
0
0.5
1
1.5
2
2.5
0 31.25 62.5 125 250 500 1000
浓度（p g/m l）
O
D值
上海尊龙凯时人生就是搏生物科技有限公司 www.hualeizs.com
4.洗板5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) 。用封板胶纸封住反应孔，室温（25～28℃）孵育60分钟。
6.洗板5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20分钟。
8.洗板5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5.8pg/ml。
2.特异性：与人的G-CSF、IL-6 、IL-12/IL-23 p40及小鼠IL-12 p70 、IL-12/IL-23 p40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. 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
上海尊龙凯时人生就是搏生物科技有限公司 www.hualeizs.com上海尊龙凯时人生就是搏生物科技有限公司 www.hualeizs.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.
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