Because the ELISA kit has many advantages such as high accuracy, high sensitivity and strong specificity, it is deeply loved by the majority of teachers and students. Now there are both domestic and imported products on the market, the number of which is dazzling, and the quality is also uneven. Now I will summarize a few selections of winning books and welcome everyone to make corrections.
1. Type of experiment
Although there are many types, they all have one common feature, which is antigen capture. The general capture format includes adsorption of antigen to microplates or capture with antibodies on microplates. In-cell ELISA and ELISPOT are two special types. In-cell ELISA requires fixation and permeabilization of cells cultured in microplates, and then in-situ detection with antibodies. ELISPOT is a bit like Western blot. First, cells are cultured in a microplate with a membrane, and then the antigen secreted by the cells is detected on the membrane to form spots. In addition, we also need to choose 96-well plates or 384-well plates for ELISA experiments according to our needs.
2. Antibody type
Both monoclonal and polyclonal antibodies can be used in ELISA kits. In fact, sometimes the two combine better. For example, for the double-antibody sandwich method, capturing with multiple antibodies and detecting with monoclonal antibodies is sometimes more helpful. The polyclonal antibody can ensure that all antigens are captured, and then the monoclonal antibody re-specifically detects the antigen with a specific epitope.
3. Cross reaction
If conflicts or cross-reactions occur between antibodies, the specificity of the entire experiment will be affected. Among them, the compatibility brought by the source of antibody is a factor of cross reaction. Although it is becoming easier to buy antibodies from designated animals, we still need to confirm whether there will be cross-reactivity problems. When using the double-antibody sandwich method for ELISA detection, the detection secondary antibody must specifically target the detection primary antibody, and cannot react with the capture antibody. If the detection secondary antibody is combined with the capture antibody, the experimental specificity will be greatly reduced. Usually we choose capture antibodies and detection of primary antibodies from different hosts to avoid the above problems.
4. Detection method
There are many ways to detect ELISA today, we can choose according to our preferences. Generally, the ELISA detects the soluble product of the enzymatic reaction. The antibody is bound to the corresponding enzyme (for example, the commonly used horseradish peroxidase HRP), and the reaction system is added with the corresponding substrate (such as 3,3-di Aminobenzidine DAB). This enzymatic reaction produces a characteristic color, which can be detected by a spectrophotometer. Alkaline phosphatase AP is also a commonly used enzyme. The enzyme can be bound to the primary antibody or to the secondary antibody, or an enzyme labeled with streptavidin can be combined with the primary antibody labeled with avidin. In addition, the results of ELISA detection also include radioactive detection, fluorescence detection, chemiluminescence or color detection.
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