Antibody accessibility
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Depending on the characteristics of the antigen studied and the method of preparation, some epitopes are not easily accessible to antibodies. The most common reasons for homologous epitope space recognition by antibodies are: â‘ the presence of other molecules, especially proteins or nucleic acids that have been bound to the antigen molecule, can compete close to the epitope; â‘¡ modification of protein antigens, especially by The phosphorylation of proteins regulated and controlled by cells; â‘¢ the problem of antigen localization in complexes as found in cell or tissue staining.
(1) Multimer interaction
One type of blockage is caused by other molecules bound to the antigen molecule. This is due to protein-protein molecule interactions. If the epitope recognized by the antibody overlaps with the interacting protein binding site in the study, no antibody-antigen binding can be detected. This can be used as a method to determine whether protein-protein molecules interact. This epitope competition can be found in almost any intermolecular interaction, but the most common is the interaction between related macromolecules, such as proteins or nucleic acids. Multimer interaction is a common problem in immunostaining, immunoprecipitation, and immunoaffinity purification, but it is rare for immunoblotting because the technology
The antigen has been completely denatured before the antibody is added.
For a specific antibody-antigen, multimer interaction is an important factor that affects the epitope recognized by the antibody, so usually it can be solved by replacing an antibody that recognizes a different epitope. If accurate information about the binding sites of different antibodies is lacking, a simple method is to use antibodies for detection. Since polyclonal antibodies usually recognize antigens at multiple sites, the binding to antigens is dominant, so polyclonal antibodies become the first choice when solving the problem of polymer interactions. The second solution to this problem is to disrupt the multimer interaction before adding the antibody. Most of the interactions between protein-DNA, protein-RNA and protein-protein molecules can be destroyed by adding 400 mmol / L or higher concentration of salt, and the structure of the antigen is also destroyed, but most antibody pairs The antigen remains active. For stronger polymer interactions, more severe conditions can be used to break the antigen-competitor relationship.
(2) Post-translational modification of protein antigens
Another factor hindering the binding of antibodies to homologous protein antigens is post-translational modification. In this case, protein phosphorylation is the most common. It can block certain monoclonal antibodies from binding to antigens. Since this is a subunit that affects proteins in cells or cell populations, usually this spatial change in antigen structure will only reduce the strength of the signal. However, in some cases, the antigen can be completely modified, which prevents the antibody from binding to the corresponding antigen. This problem can be solved by replacing the antibody or removing the protein antigen with an enzyme to remove the modification. For example, in the case of protein antigen phosphorylation, it can be treated with phosphatase.
(3) Positioning
The third type of problem with antibody accessibility is caused by antigen localization, such as immunostaining. If the antigen is embedded in a complex structure, or due to a barrier cell structure, the physical properties of the antibody are not easy to bind to the antigen, which is a unique problem in the staining of cells or tissues.
Solving such problems is difficult and needs to be treated separately, because the mechanism that prevents antibodies from reaching the antigen in each case is different, and each needs to be considered as a separate problem. Various methods to address the accessibility of these antibodies involve the use of certain physical means to destroy the interfering structure. The two most commonly used methods are partial protein solubilization and microwave treatment. Partial protein solubilization purposefully removes the inhibitory structure so that the target antigen is not affected. To make protein solubilization more effective, the amount of protease and incubation time need to be adjusted. The immobilization method can provide some help, because physically many areas of the antigen are bound to adjacent structures through cross-linking, so even if protein dissolution splicing of an antigen occurs, at least part of the antigen is still connected to other local structures . Nonetheless, only achieving spatial accessibility is an effective method.
The second commonly used method to achieve accessibility is to use microwaves to process the sample, and to partially denature the cell structure to achieve the goal. However, it is necessary to carefully adjust the time and intensity of microwave treatment in order to make the antibody accessible, but without destroying the antigen or identifiable cell morphology.
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