Basic principles of antigen peptide selection
As much as possible on the surface of the protein
Ensure that this sequence does not form α-helix
N,C Peptides at the ends are better than peptides in the middle
Avoid repeats or sequences close to repeats within the protein
Avoid peptides with strong homology
Cross-linking can be done at N, C At both ends, the selection is based on the fact that the cross-linking is at the end that is less important for the production of antibodies.
There cannot be too many in the sequence Pro, But there are one or two Pro It has the advantage that it can make the peptide chain structure relatively stable, which is beneficial to the production of specific antibodies.
In order to achieve the best results in producing antibodies, it is necessary to carefully design the antigen peptide.
The design should meet a basic condition: During the immunization process, the antigen will not produce an excessive immune response, but at the same time, it can produce antibodies with the ability to bind to the protein of interest.
Although antigen design is a very complex topic with many details that need to be paid attention to, based on our accumulated experience, there are several key basic design principles that can be provided for your reference.:
1, Determine the purpose of the antibody
When starting a new research project, it is necessary to clarify some basic properties of the protein of interest, especially if knowing the structure of the protein will be of great help in selecting recognition regions that the antibody can easily access and recognize. However, in the absence of such precise structural information (which is mostly the case), understanding the purpose of the study affects the strategy for peptide design. For example: If the research focus is on different regions of the protein, e.g. C terminal or N end, or the protein in a specific state, such as phosphorylation, etc., then the polypeptide designed according to the required sequence and the corresponding antibody generated should not have much difficulty in application. However, the conformation of the protein will affect the interaction between the antibody and its recognition region. A possible problem in this situation is that if in the folded protein the recognition region is hidden inside the protein, the antibody will not be able to access this region. (Cannot interact).
2, Identification area selection principles
Generally speaking, the most ideal antigenic recognition region should be hydrophilic, located on the protein surface, and structurally deformable. Because in most natural environments, hydrophilic regions tend to be concentrated on the protein surface, while hydrophobic regions are often wrapped inside the protein. For the same reason, antibodies can only interact with recognition regions found on the protein surface. When these recognition regions have sufficient structural deformability to move to positions where the antibody can contact, they will have a high affinity with the antibody.
3, Continuous and discontinuous recognition areas
A continuous region refers to a recognition region composed of continuous amino acid sequences. Most antibodies are directed against continuous recognition regions. The ability of antibodies to bind to such regions with high affinity indicates that this sequence is not within the protein. The discontinuous recognition region represents a polypeptide sequence with a certain fold, or the recognition region of an antibody that connects two separate polypeptides together. In some cases, antibodies against such discontinuous recognition regions can also be produced, but the antigen polypeptide used for immunization must have a secondary structure similar to the discontinuous recognition region, and the length of the sequence needs to meet relevant requirements.
4, Basic advice
To avoid the risk of the recognition region being hidden inside the protein, we usually recommend choosing a N, C corresponding antibodies produced at both ends. Because in intact proteins, N, C Both ends are usually exposed on the protein surface. However, it is important to note that membrane proteins C The end is too hydrophobic and is not suitable as an antigen.
5, Sequence length
Generally, we recommend that the sequence length of the antigen polypeptide be between 8-20 amino acid residues. If it is too short, there is a risk that the peptide will be too special and the affinity (binding ability) between the produced antibody and the natural protein will not be strong enough. Similarly, if the sequence length exceeds 20, it is possible to introduce secondary structures and the produced antibodies may lose specificity. Moreover, the longer the peptide chain, the more difficult it is to synthesize, making it difficult to obtain high-purity products.
6, Choice of carrier protein cross-linking
Basic principles: Add the carrier protein to the end far away from the antibody recognition region, which is not included in the sequence. Cys In the case of N or C end plus Cys The preferred method for cross-linking.
7, Commonly used analysis software
MacVecfor TM; DAN star TM; PC-Gene TM