The creation of antigens is essential for the production of monoclonal or polyclonal antibodies.For the antigens which are difficult to acquire, small molecular weight and high homology, we can consider the custom peptide method to synthetic the antigens.
Peptide antigen VS Protein antigen
|Peptide antigen||Protein antigen|
• Western Blot
• PTM Detection
• High homology between target protein with other proteins
• Epitopes understanding
• Western Blot
• Functional assay
• Low homology between target protein with other proteins
• Accurate recognition of epitopes
• Less cross-reaction
• Flexible antigen area selection
• Easy synthesis, low cost, short delivery time
• Produce antibodies at multiple epitopes
• Immunogen protein is positive for WB test
• Need coupling with carrier protein
• Linear epitope recognition
• Higher nonspecific cross-reaction
• The production is relatively long and the price is high.
The core of peptide antigen scheme is peptide selection and epitope design. Aurora has over ten years of experience in the synthesis of peptide antigens. We will design and produce the best antigen for you by combining the use of antibody and considering the factors of homology, hydrophilicity, molecular weight, structure, purity and length.
Antigen design theory
Antigen refers to a substance that can stimulate human or animal body to produce antibodies or primed lymphocytes, and can react specifically with these products in vivo or in vitro. The basic ability of antigen include antigenicity and immunogenicity. Antigenicity refers to the ability of antigen to specifically combine with antibody or primed lymphocyte which induced by antigen.The strength of antigenicity is closely related to the molecular size, chemical composition and structure of antigenic determinants .Immunogenicity refers to the ability to stimulate the body to form specific antibodies or primed lymphocytes.Excellent peptide antigens can stimulate specific immune cells, activate, proliferate and differentiate immune cells, and finally produce specific antibodies and primed lymphocytes.
The area on the surface of a protein that makes the immune system produce antibodies is called an antigenic determinant.A natural antigen can have multiple determinants. The larger the antigen molecule, the more the amount of determinants.The antigenic determinants with well-defined structure are called epitopes.Based on the statistical data of amino acids (surface accessibility,hydrophilicity,flexibility)and the prediction of protein senior structure(β-bend, etc) the area of antigenic determinant can be calculated in a certain extent.
Design philosophy of peptide antigen
it is essential to design the antigen peptide thoroughly to acquire optimum effect of the antibody. The design should meet a main condition: the antigen will not produce too strong immune reaction but also can produce antibodies with binding ability to the protein of interest in the process of immunity.Based Omizzur ’s years experience here are several key basic design principles to provide you with reference:
The designed peptide sequence has no homology with the protein sequence of the immunized host, nor with other protein sequences of its own species.We achieve sequence alignment by NCBI BLAST.
All proteins expose the hydrophilic region to the surface and the hydrophobic region to the inside of the them.Therefore, the designed peptide must have good hydrophilicity to be better soluble in the water phase and to stimulate the immune reaction.(Hydrophilicity can be predicted by our antigenic determinant prediction tool)
3. Peptide terminal
In whole proteins, both N and C terminal are usually exposed to the protein surface,However the C-terminal is hydrophobic to some degree, so we can choose the N-terminal, which makes it easier to produce antibodies.
4. Peptide structure
The sequences do not form a-helix and duplicate segments internal .
5. Length and purity
In order to prevent protein hydrolysis and ensure epitope coverage, peptide antigens are generally designed with 8-20 amino acids.If the length is too short, the affinity between the antibody and the natural protein is not strong enough,and if too long,secondary structure may happens.
The purity of ≥ 85% of peptide used to produce antibody can be appropriate.
6. Sequence optimization
Sequence contains such as Asp, Tyr and Phe, which are preferred by MHC molecules(major histocompatibility complex).Avoid amino acids that are easy to glycosylation:Asn,Ser.Thr and phosphorylated amino acids.The proper Pro is good for stability of peptide sequences.
7. Carrier linker
Immunogenicity can be acquired by coupling peptide antigen with carriers such as macromolecular proteins.Generally, a linker arm is added to the end of the peptide chain which has minimum influence on antibody production , to facilitate the coupling of the peptide with the carrier.Adding Cys at the N or C terminal which without Cys in the sequence is the preferred method.
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