Description:
1. Intended Use
The GenWay Candida albicans IgM Antibody ELISA Test Kit has been designed for the the detection and the quantitative determination of specific IgM antibodies against Candida albicans 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
Invasive candidal mycosis (synonyms: candidosis, candidiasis) account for more than 80% of the cases of deep-seated mycoses in Germany. This incidence is similar to those of other industrial countries. The main causative agent is the yeast Candida albicans, which is the only fungal species belonging to the normal microflora. Candida albicans has a low pathogenic potential, which rarely allows it to infect a healthy host. Rather, in immunosuppressed individuals it may cause several diseases as an opportunistic pathogen. In candidal mycosis three types of disease can be distinguished. First, Candida albicans can infect the skin and the mucous membranes of the outer genitals, mouth, respiratory tract and digestion tract. Second, skin and mucous membranes may be infected, encouraged by factors like pregnancy, diabetes mellitus, immunodeficiency and therapies with cytostatics or antibiotics. Third, the infestation of the inner organs, for example the lungs in a Candida sepsis can cause death in immunosuppressed individuals with a cellular immunodeficiency. Serological tests are frequently used to ascertain the clinical significance of Candida spp. isolates. Rising titers of lgG antibodies to Candida albicans may reflect invasive candidiasis in immunocompetent individuals. The detection of IgA and IgM antibodies is important to identify a fresh infection. Immunosuppressed individuals often fail to produce antibodies, so a negative antibody test does not necessarily rule out the disease. In this case the use of an antigen detection test is recommended. Besides the detection of antibodies and antigen, the site of manifestation, the number of colony-forming units, and the concomitant use of antimycotics must be considered.
3. Principle of the Test
The GenWay Candida albicans IgM antibody test kit is based on the principle of the enzyme immunoassay (EIA). Candida albicans 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 IgM antibodies of the serum and the immobilized Candida albicans 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-IgM 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 IgM antibodies is directly proportional to the intensity of the color.
Additional Information:
| Name | Candida albicans IgM ELISA |
|---|---|
| Related Product Names | Candida albicans IgM ELISA |
| Applications | Enzyme immunoassay based on microtiter plate for the detection and quantitative determination of human IgM antibodies against Candida albicans 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。
