Keep in mind that the immune system stimulates the peptide-protein as a whole, and the antibodies produced are specific to the peptide, both to the linker and to the carrier protein. The most common carrier proteins are as follows:
KLH (Keyhole Limpet Hemocyanin), a hemocyanin, is a free blue respiratory pigment found in the hemolymph of certain mollusks, arthropods (spiders and beetles). Hemoglobin contains two copper ions directly linked to the polypeptide chain. Similar to iron-containing hemoglobin, it is easy to combine with oxygen and easily dissociates from oxygen. It is the only known copper protein that can reversibly bind to oxygen. Cyan, white when restored. Its molecular weight is 450,000 to 130,000. Because KLH is more immunogenic than BSA, it is the most commonly used carrier protein.
BSA (BovineSerumAlbumin), bovine serum albumin, belongs to the most stable and soluble albumin. Its molecular weight is 67x103 Da (containing 59 Lys). Approximately 30-35 major amino groups are available for conjugation with the linker, making BSA a very popular weak antigen compound carrier protein.
The disadvantage of BSA is that it is used as a blocking agent in many experiments. If the antiserum of the peptide-BSA conjugate is used in such assays, false positives usually occur because these sera contain anti-BSA. antibody.
OVA (Ovalbumin), chicken egg albumin, has a molecular weight of 45 x 103 Da. It serves as a second carrier protein to verify whether the antibody specifically targets only the polypeptide and not the carrier protein (such as BSA).
The thiol modification (via the side chain of Cys) is used to conjugate with KLH, BSA or OVA, and all functional groups carrying a thiol reaction can be modified to effect a conjugation reaction. The most common ones are the following:
Iodoacetamide
Maleimide
Alkylhalide
KLH/BSA/Ovalbumin etc coupling
The small peptide/hapten must be coupled to a carrier protein (KLH, BSA, Ova) to obtain a highly efficient antibody. In general, the conditions under which a polypeptide can be coupled to a protein are as follows:
1. There is a free amino or carboxyl group.
2. -SH on cysteine ​​can also be coupled with carrier protein. Currently, our company provides high quality coupled carrier protein (KLH, BSA, OVA).
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