Description:
1. Intended Use
The GenWay Adenovirus IgG Antibody ELISA Test Kit has been designed for the the detection and the quantitative determination of specific IgG antibodies against Adenovirus in serum and plasma. Further applications in other body fluids are possible and can be requested from the Technical Service of GenWay. This assay is intended for research use only.
2. General Information
The adenovirus is an ubiquitous pathogen of humans and animals. Adenoviruses are characterized by location inside the cell nucleus, common complement-fixing antigens and marked stability to environmental effects. Adenoviruses are endemic in all populations throughout the year. The infection is spread both through the aerial-droplet route and the routes characteristic for intestinal infections. The incubation period is between five and seven days. Adenoviruses mainly infest respiratory and intestinal mucosa, but also the cornea. They are accumulated in the epithelial cells and regional lymph nodes. Adenoviruses cause the widest variety of illnesses of the known respiratory viruses. The adenovirus infection is the most frequently caused viral disease of the respiratory tract among preschool children (types 1 - 5 and 7). Acute diseases of the upper respiratory tract occur predominantly. Pneumonia is the most severe form of adenoviral infection occurring mostly in infants below the age of one. Adenoviruses also cause outbreaks of swimmingpool-associated pharyngoconjunctival fever in the summer and epidemics of kerato-conjunctivitis of both children and adults. The intestinal form of adenoviral infection occurs mostly in children below the age of one. An acute adenoviral infection can be detected by virus isolation and/or serology. The serologic tests are particularly important because they document actual infection in the individual and can be applied to large-scale epidemiologic investigations. The CF and ELISA tests measure predominantly the antibodies directed against the group-specific determinants on the hexon component. The recommended tests for measuring type-specific antibodies are hemagglutinin inhibition and serum neutralization. The type-specific antigenic determinants of adenoviruses are located at the fibers on the capsid. Because of the ubiquity of the adenoviruses and numerous cross-reactions between related serotypes, seroconversion involving a fourfold or greater rise in antibody infection is necessary to document infection. IgG is the predominant antibody class measured in the serologic tests.
3. Principle of the Test
The GenWay Adenovirus IgG antibody test kit is based on the principle of the enzyme immunoassay (EIA). Adenovirus antigen is bound on the surface of the microtiter strips. Diluted sample serum or ready-to-use standards are pipetted into the wells of the microtiter plate. A binding between the IgG antibodies of the serum and the immobilized Adenovirus antigen takes place. After a one hour incubation at room temperature, the plate is rinsed with diluted wash solution, in order to remove unbound material. Then ready-to-use anti-human-IgG peroxidase conjugate is added and incubated for 30 minutes. After a further washing step, the substrate (TMB) solution is pipetted and incubated for 20 minutes, inducing the development of a blue dye in the wells. The color development is terminated by the addition of a stop solution, which changes the color from blue to yellow. The resulting dye is measured spectrophotometrically at the wavelength of 450 nm. The concentration of the IgG antibodies is directly proportional to the intensity of the color.
Additional Information:
| Name | Adenovirus IgG ELISA |
|---|---|
| Related Product Names | Adenovirus IgG ELISA |
| Applications | Enzyme immunoassay based on microtiter plate for the detection and quantitative determination of human IgG antibodies against Adenovirus in serum and plasma |
| Intended Use | Research Use Only |
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2.加大辣根过氧化物酶标记的链霉亲和素；
我现在用商品化的试剂盒进行夹心法ELISA测定某抗体的浓度，盒子给定了4个标准品，标准品抗体浓度为70000~1000单位，测定步骤中要求对样品进行1:1000稀释后测定。
但是有的样本按照1:1000稀释后，最终OD值大于70000浓度的标准品的OD值，然后使用ELISACALC软件进行四参数拟合，超过了标准曲线的范围，就算不出来这个样品的浓度，但是如果在1000-70000之间的OD值就可以算出相应浓度。请教大神们，接下来我是否可以对样品进行1:2000,1:4000浓度稀释，算出结果后再×2、×4，算作此样品的浓度呢？还是直接给一个＞70000的定性结果？或者可以有能算出来的其他软件？
Ps试剂盒中提及Dilutionlinearity（稀释线性？）为141%，这个是什么意思呢？
多谢！！
请大家帮帮忙，第一步加完抗原后必须封闭吗
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。
ELISA双抗体夹心法(enzyme linked immunosorbent assay——sandwich technique)的原理是将特异性抗体结合到固相载体上形成固相抗体，然后和待检血清中的相应抗原结合形成免疫复合物，洗涤后再加酶标记抗体，与免疫复合物中抗原结合形成酶标抗体-抗原-固相抗体复合物，加底物显色，判断抗原含量。
生物帮有相关介绍。编码RNA http://doc.bio1000.com/show-3399.html
（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。
