Peptide-carrier protein coupling is often used to prepare anti-polypeptide antibodies. A single peptide is usually too small to provoke an adequate immune response, while carrier proteins with many antigenic epitopes are beneficial to stimulate helper T cells and further induce B cell immune response.
Remember, the immune system uses peptide-protein as a whole to provoke an immune response, so antibodies are made against peptides, linkers, and carrier proteins. The most common carrier proteins are as follows:
1. Keyhole Limpet Hemocyanin (KLH), or haemocyanin, is a free blue respiratory pigment found in haemolymph of certain mollusks, arthropods (spiders and beetles). Hemocyanin contains two copper ions directly connected to the polypeptide chain, which is similar to iron-containing hemoglobin. It is easy to bind with oxygen, and also easy to dissociate with oxygen. It is the only known copper protein that can be reversibly bound with oxygen, appearing green in oxidation and white in reduction. Its molecular weight is 450000 ~ 130 000. KLH is the most commonly selected carrier protein due to its higher immunogenicity than BSA.
2. BSA (Bovine Serum Albumin), 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 are available for conjugating with the linker, making BSA a popular carrier protein for weak antigens.
The disadvantage of BSA is that it is used as a blocking agent in many experiments. If antiserums of peptide-BSA conjugate are used in such assays, they will often show false positives because they contain antibodies against BSA.
3, OVA (Ovalbumin), namely chicken egg albumin, molecular weight is 45 x 103 Da. It can be used as a second carrier protein to verify that the antibody specifically targets only the peptide and not the carrier protein (e.g., BSA).
In general, peptides can be coupled to proteins under the following conditions:
◆ Mercapto (-SH) on Cys can carry out coupling reaction (recommended)
◆ Or have a free amino or carboxyl group