The peptide antigen synthesized by chemical is a small molecule, and it is difficult to have good antigenicity. It can only induce a weak immune response in animals. Therefore, it is very important to couple peptide with the carrier protein. The carrier protein contains many antigenic determinants, which can stimulate T-cells and induce B-cell response.

One aspect that is often neglected in designing synthetic peptides is how to couple peptides to carrier proteins. For example, the N-terminal sequence needs to be coupled through the C-terminal amino acid, while the C-terminal sequence needs to be coupled through the N-terminal amino acid.All coupling methods should be to couple peptides to carrier proteins through carboxyl or amino terminal residues. No more than one residue of the selected sequence can participate in the selected coupling chemical reaction. If there are multiple reaction sites, we can consider shortening the peptide sequence, or selecting the peptide with all reaction sites at one end. For internal sequences, the ends far away from the predicted antigen sites are usually used for coupling, which can avoid possible shielding problems

The immune system reacts to the peptide carrier protein as a whole, that is to say, part of the immune response is aimed at coupling polypeptides, some is against the intermediate linker, and the other part is against the carrier protein. When ELISA is used for screening, it is recommended to use peptide polymers coupled with different carrier proteins. This is not necessary if the ELISA is performed on a porous plate coated with uncoupled peptides.The most common carrier proteins are as follows:

Common carrier protein coupled with peptide

1. BSA

BSA (bovine serum albumin) is the most stable and soluble albumin. Its molecular weight is 67 x 103 Da (containing 59 Lys). About 30-35 major amino groups can be used for conjugation with linkers, making BSA a popular carrier protein of weak antigenic compounds.

The disadvantage of BSA is that it is used as a blocking agent in many experiments. If the antiserum of peptide BSA conjugate is used in such detection and analysis, false positive results will usually appear because these serum contain anti BSA antibodies

2. KLH

KLH (Keyhole Limpet Hemocyanin), a blood blue pigment, is a free animal pigment found in hemolymph of animal mollusks, arthropods (spiders and beetles). KLH contains two copper ions directly linked to the polypeptide chain. Similar to hemoglobin containing iron, KLH is easy to combine with oxygen and dissociate easily with oxygen. KLH is the only known copper protein that can bind to oxygen reversibly. It is green when oxidized and white when reduced. Its molecular weight is 450000-130000. KLH is the most commonly used carrier protein because of its higher immunogenicity than BSA.

3. OVA

OVA (Ovalbumin) is a chicken egg white protein with a molecular weight of 45 x 103 da. It can be used as a second carrier protein to verify whether the antibody specifically targets the peptide rather than the carrier protein (such as BSA).

Because the antiserum produced by peptide coupled with BSA also contains antibodies directed against BSA, which may result in false positive results at one time. Although KLH is large and antigenic, it may precipitate during the coupling process, so it is sometimes difficult to deal with. Ovalbumin (OVA) is another carrier protein that can be used. Ova is a good choice as a second carrier when the antibody is to be tested only for polypeptides and not for carrier proteins. Rabbit serum albumin (BSA) can be used as a carrier to minimize the antibody anti carrier reaction. Rabbits immunized with RSA coupons are less likely to produce antibodies against the vector because RSA is the rabbit's own protein. If the injected animals are not rabbits, RSA will not be considered as autologous protein

All functional groups carrying thiol group can be used for conjugation reaction after modification. The most common ones are the following:Iodoacetamide,Maleimide,Alkylhalide.

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