Description:
Product Name: Interleukin-6
Category: Immunology Markers
Sample Type: Serum
Sensitivity: 2 pg/mL
Description: Immunoenzymetric assay for the in vitro quantitative measurement of human Interleukin-6 (IL-6) in serum.
# of Samples: 1X96 Assays
Intended Use: Immunoenzymetric assay for the in vitro quantitative measurement of human interleukin-6 (IL-6) in serum.
Background: A. Biological activities
Human Interleukin 6 (IL-6) is a 184 A.A. polypeptide with potential O and N-glycosylation sites, and a significant homology with G-CSF. It is produced by various cells, including T- and B-cells, monocytes, fibroblasts, keratinocytes, endothelial cells, mesangial cells, astrocytes, bone marrow stroma cells and several tumor cells. It regulates the growth and differentiation of various cell types with major activities on the immune system, hematopoïesis, and inflammation. These multiple actions are integrated within a complex cytokine network, where several cytokines induce (IL-1, TNF, PDGF, IFNs,...) or are induced by IL-6 and the final effects result from either synergistic or antagonistic activities between IL-6 and the other cytokines (IL-1, IL-2, IL-4, IL-5,IFN¬, IL-3, GM-CSF, M-CSF,CSF,...). IL-6 induces final maturation of Bcells into antibody producing cells and is a potent growth factor for myeloma/plasmacytoma cells. It (co-) stimulates T-cell growth and cytotoxic T-cell differentiation. It promotes megakaryocyte development and synergizes with other cytokines to stimulate multipotent hematopoïetic progenitors. It can also induce differentiation and growth inhibition of some leukemia -or non hematopoïetic tumoral cell lines. IL-6 is also a major inducer of the acute phase reactions in response to inflammation or tissue injury. Along with IL-1 and TNF, it induces the synthesis of acute phase proteins (APP) by hepatocytes, each cytokine or combination of cytokines showing a preferential pattern of APP production. IL-6 also interacts with the neuroendocrine system, e.g. by inducing ACTH production. Thus, IL-6 is a pleiotropic cytokine with multiple endocrine, paracrine and possibly autocrine activities in various tissues.
Principles of the method: The GenWay IL-6-EASIA is a solid phase Enzyme Amplified Sensitivity Immunoassay performed on microtiterplate. The assay uses monoclonal antibodies (MAbs) directed against distinct epitopes of IL-6. Calibrators and samples react with the capture monoclonal antibody (MAb 1) coated on microtiter well and with a monoclonal antibody (MAb 2) labelled with horseradish peroxidase (HRP). After an incubation period allowing the formation of a sandwich: coated MAb 1 - human IL-6 - MAb 2 - HRP, the microtiterplate is washed to remove unbound enzyme labelled antibody. Bound enzymelabelled antibody is measured through a chromogenic reaction. Chromogenic solution (TMB) is added and incubated. The reaction is stopped with the addition of Stop Solution and the microtiterplate is then read at the appropriate wavelength. The amount of substrate turnover is determined colourimetrically by measuring the absorbance, which is proportional to the IL-6 concentration. A calibration curve is plotted and IL-6 concentration in samples is determined by interpolation from the calibration curve. The use of the EASIA reader (linearity up to 3 OD units) and a sophisticated data reduction method (polychromatic data reduction) result in a high sensitivity in the low range and in an extended calibration range.
Storage and Stability: Before opening or reconstitution, all kits components are stable until the expiry date, indicated on the vial label, if kept at 2 to 8C.
Unused strips must be stored, at 2-8C, in a sealed bag containing a desiccant until expiration date.
After reconstitution, calibrators, controls and specimen Diluent are stable for 4 days at 2 to 8C. For longer storage periods, aliquots should be made and kept at -20C for maximum 2 months. Avoid successive freeze thaw cycles.
The concentrated Wash Solution is stable at room temperature until expiration date.
Freshly prepared WorkingWash solution should be used on the same day.
After its first use, the conjugate is stable until expiry date, if kept in the original well-closed vial at 2 to 8C.
Alterations in physical appearance of kit reagents may indicate instability or deterioration.
Sensitivity and Specificity: No significant cross-reaction was observed in presence of 50 ng of IL-1", IL-1#, IL-2, IL-3, IL-4, IL-7, IL-8, IL-10, GM-CSF, IFN-", IFN- , LIF, MIP-1", MIP- 1#, MCP-1, OSM, RANTES, TGF-#, TNF-" and TNF-#. A very tenuous crossreaction (0.06%) is observed with G-CSF.
Product Name: Interleukin-6
Function: IL-6 is a cytokine with a wide variety of biological functions: it plays an essential role in the final differentiation of B-cells into Ig-secreting cells, it induces myeloma and plasmacytoma growth, it induces nerve cells differentiation, in hepatocytes it induces acute phase reactants.
Subcellular Location: Secreted.
Ptm: N- and O-glycosylated.
Polymorphism: Genetic variations in IL6 may be correlated with bone mineral density (BMD). Low BMD is a risk factor for osteoporotic fracture. Osteoporosis is characterized by reduced bone mineral density, disrutption of bone microarchitecture, and the alteration of the amount and variety of non-collagenous proteins in bone. Osteoporotic bones are more at risk of fracture.
Disease: Genetic variations in IL6 are associated with susceptibility to systemic juvenile rheumatoid arthritis [MIM:604302]. Systemic juvenile rheumatoid arthritis is juvenile chronic arthritis associated with severe, debilitating, extraarticular features, and occasionally fatal complications. Despite medical treatment, many children still experience early joint destruction, necessitating surgical replacement.
Disease: At least 1 polymorphism in the IL6 gene renders HIV-infected men susceptible to Kaposi sarcoma [MIM:148000]. Kaposi sarcoma is a sarcoma of spindle cells mixed with angiomatous tissue. A relatively rare malignant skin tumor that results in multifocal purplish coloured papules or plaques that eventually form nodules. Most commonly seen in patients who suffer from AIDS.
Similarity: Belongs to the IL-6 superfamily.
Additional Information:
| Name | Interleukin-6 ELISA Kit |
|---|---|
| Related Product Names | Interleukin-6 ELISA Kit; IL-6; B-cell stimulatory factor 2; BSF-2; Interferon beta-2; Hybridoma growth factor; CTL differentiation factor; CDF IFNB2Interleukin-6IL-6 |
| NCBI Acc Number | NP_000591.1 |
| Molecular Weight | 23718 |
| Swiss Prot Number | P05231 |
| Intended Use | Research Use Only |
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下图为ELISA原理图,夹心法即Sandwich ELISA,竞争法就是Competitive ELISA
向左转|向右转
(2)封闭:将已包被的板用洗涤液洗涤2次,每孔加入120 μL封闭液,封闭一段时间,取出,甩干备用。
(3)竞争反应:在制备好的板中每孔加入50 μL系列浓度的标准品溶液和50 μL稀释好的酶标抗体溶液,孵育一段时间。
(4)洗板:用洗涤液洗涤5次。
(5)显色反应:每孔加入显色溶液100 μL,孵育一段时间。
(6)终止:每孔各加入终止液,在酶标仪上测定各孔的吸光值。
1.包被:用0.05MPH9.牰碳酸盐包被缓冲液将抗体稀释至蛋白质含量为1~10μg/ml。在每个聚苯乙烯板的反应孔中加0.1ml,4℃ 过夜。次日,弃去孔内溶液,用洗涤缓冲液洗3次,每次3分钟。(简称洗涤,下同)。
2. 加样:加一定稀释的待检样品0.1ml于上述已包被之反应孔中,置37℃ 孵育1小时。然后洗涤。(同时做空白孔,阴性对照孔及阳性对照孔)。
3. 加酶标抗体:于各反应孔中,加入新鲜稀释的酶标抗体(经滴定后的稀释度)0.1ml。37℃ 孵育0.5~1小时,洗涤。
4. 加底物液显色:于各反应孔中加入临时配制的TMB底物溶液0.1ml,37℃ 10~30分钟。
5. 终止反应:于各反应孔中加入2M硫酸0.05ml。
6. 结果判定:可于白色背景上,直接用肉眼观察结果:反应孔内颜色越深,阳性程度越强,阴性反应为无色或极浅,依据所呈颜色的深浅,以“+”、“-”号表示。也可测OD值:在ELISA检测仪上,于450nm(若以ABTS显色,则410nm)处,以空白对照孔调零后测各孔OD值,若大于规定的阴性对照OD值的2.1倍,即为阳性。
间接法
1.用包被缓冲液将已知抗原稀释至1~10μg/ml,每孔加0.1ml,4℃过夜;
2.次日洗涤3次;
3.加一定稀释的待检样品(未知抗体)0.1ml于上述已包被之反应孔中,置37℃孵育1小时,洗涤;
4.(同时做空白、阴性及阳性孔对照)于反应孔中,加入新鲜稀释的酶标第二抗体(抗抗体)0.1ml;
5.37℃孵育35-60分钟,洗涤;
6.’最后一遍用DDW洗涤。
其余步骤同“双抗体夹心法”的4、5、6。
1.标准曲线的标准品是否一定要梯度稀释,为什么?我试过非梯度稀释的,也可以达到线性R2=0.99.
2.我用了CurveExpert做标曲,自动搜索后发现有10种提供的方程,各种形式的,其中一个十分适合我的实验结果(LogisticModel),而其他的感觉又不适合,因为结果常常为负值。这又是为啥捏?
3.实验的酶标仪最大OD值可以测到4,如果我的测量结果在1.3,是否像其他人所说的>1了就不准确了。
4.利用夹心法进行定量分析是否一定要使用线性方程?
不好意思啊,一下问了这么多问题,最近做了一个月的ELISA,完全摸不清头脑啊。谢谢各位了
后采用豆粉浸提12000g离心取上清的方式,发现10的5、6、7、8次方稀释度所测OD值随着稀释度的升高而增大,比如一个样品的OD值5次方为0.2558次方为0.349;
刚接触相关知识,试验任务较重,心中疑问较大,恳请各位高手及时指正,诚谢!
表面抗体是双抗原夹心法
E抗体和核心抗体是竞争抑制法或间接法
如果用双抗体夹心法,第二个抗体可以用多抗,这样保证你的抗原会被结合,多抗可以直接带HRP标记或者可以用HRP标记二抗去和多抗结合。
目前,我用该蛋白的鼠源性单抗来包被ELISA板子,5%BSA37度封闭二小时后,洗涤四次后将板子分三个组加入抗原:其中一个组中加入的是我重组的蛋白,另一个组为用其它方法证实含该蛋白的人体内血清,最后一个组加入PBS。37度一小时后洗涤四次后每孔加入我制备的兔源性多抗。37度一小时洗涤六次后我再加入酶标记的抗兔的抗体37度40分钟后洗涤六次后TMB显色。
问题就在我显色后所有的孔都是阳性,其中以抗原为我的重组蛋白组与PBS组OD值最高,两组都可以达到1.8以上,而加入的抗原为人体血清组低,为0.6-1.0不等。
后面我优化条件,封闭液用过5%的脱脂奶粉,2%的BSA,包被的单抗浓度摸了梯度,加入的多抗也摸了梯度,酶标的抗体也摸了梯度。结果都没有改变,连趋势每次都是一样以重组蛋白组与PBS组OD最高。
今天我还试着把抗原换成其它蛋白也做出了强阳性?
请教各位高手,我的ELISA该如何往下面做了啊,是不是我的抗体本身有问题呢?
对了,我包被用的单抗是用GE公司的预装柱纯化的,很纯,在考染中是看不到一条杂带的。

