The molecular structure of antigen that can cause the body to produce antibodies is called antigenic determinant, also known as antigenic epitope. An antigen can have many different antigenic determinants, so the body can also produce many different antibodies. Monoclonal antibody is a highly uniform antibody produced by a single B cell clone, which only recognizes a specific epitope. The antibody produced by multiple B lymphocyte clones, stimulated by multiple epitopes and able to bind to multiple epitopes is polyclonal antibody. From a certain point of view, polyclonal antibody is a mixture of several monoclonal antibodies.
The hybridoma cells were obtained by cell fusion between B lymphocytes treated with specific antigen and myeloma cells. After HAT culture medium screening and ELISA assay, positive clones were obtained. Finally, the cells were cultured or injected into the abdominal cavity of animals (usually balb/c mice) for ascites culture, and the supernatant/ascites was collected and purified to obtain monoclonal antibodies. However, the preparation of polyclonal antibody is less complicated than that of monoclonal antibody. Only the antigen (the higher the purity, the better) needs to be directly injected into the animal body for immunization. After 3-4 times of immunization, after the titer of the polyclonal antibody is qualified by ELISA, the blood is collected and centrifuged to obtain the supernatant. After purification, the polyclonal antibody can be obtained. Therefore, the preparation period of polyclonal antibody is shorter than that of monoclonal antibody, and the first preparation price is lower than that of monoclonal antibody.
Monoantibodies and polyclonal antibodies have their own distinct characteristics and advantages. Monoclonal antibodies have high specificity, and once successfully prepared, completely consistent antibodies can be produced forever, so its specificity can be comprehensively and systematically verified. However, if the identified epitope is destroyed, the experimental results will be greatly affected, which is one of the shortcomings of monoclonal antibody. However, the specificity of polyclonal antibody is poor. Even if the same antigen is used to prepare polyclonal antibody, there will be differences among different batches, so there are great limitations in specificity and consistency. Therefore, when polyclonal antibody is used for immunoassay, it is easier to cause background, for example, there are mixed bands in WB, dark background in IHC and so on. Although there is still the problem of cross-reaction *, because the polyclonal antibody recognizes multiple epitopes, even if a few epitopes are destroyed or antigen conformation changes, the experimental results will not be affected. Under the same conditions, using polyclonal antibody can improve the sensitivity of detection, and it is easier to detect proteins with low abundance.
If the specificity of the antibody is high, the dosage is large, or the consistent antibody needs to be used for a long time, and the application requirements of the prepared antibody are high (WB/IP/IF/ICC, etc.), you can choose to prepare monoclonal antibody. If the specificity of the antibody is not high, the detection experiment of precipitation and agglutination reaction or only ELISA test is needed, and polyclonal antibody can be prepared.