The generation of custom antibodies can be accomplished by using one of two primary approaches: full length protein and short peptides, but synthetic peptide antigens are often too small to generate significant immune responses on their own.To overcome the bottleneck, synthetic peptides are conjugated to bigger carrier proteins, which containing many epitopes help to stimulate T cells and induce B-cell response. Some of the most common carrier proteins include:
KLH is a large aggregating protein and has a MW that ranges from 4*105 to 1*107 Da. One of the advantages of KLH is that it does not interfere with ELISA or western blotting experiments because it is not used as a blocking reagent. Besides, KLH shows high immunogenicity compared to that of BSA. As shown in figure 1, PEG and PCB were conjugated to five different protein: murine serum albumin (MSA), BSA, KLH, beta‐glucosidase, or OVA, and the KLH shown extremely high immunogenicity in mammalian hosts. Due to its size and structure, the solubility in water of KLH is limited, giving solutions and mixtures a cloudy appearance. However, this does not affect immunogenicity and the turbid solution can be used for immunizations.
Fig1. Titers of protein-specific IgM (a) and IgG (b) after four weekly SC. (subcutaneous) administrations of MSA, BSA, OVA, beta-gluocosidase, and KLH in C57BL/6J mice were measured by ELISA tests. c) Illustrative scheme of proteins and polymers used for conjugation (Li, Bowen, et al.)
BSA (Bovine Serum Albumin)
BSA is a plasma protein derived from cattle, with a MW of 7 × 104 Da. Because BSA is smaller, it’s more water soluble than KLH. However, a disadvantage of BSA is that it is used in many experiments as a blocking buffer reagent. If antisera against peptide-BSA conjugates are used in antibody-based assays, false positives are common because the sera used also contain antibodies against BSA.
OVA is the most abundant protein in hen's egg white and the MW is 4.5 × 104 Da. It is often used as a second carrier protein to confirm that antibodies are specific for the peptide rather than the carrier protein such as BSA.
The carrier proteins above can be conjugated to peptides at the N-terminal, C-terminal or internal residues. We recommend binding carrier protein to the N- or C-terminus of the peptide, in order to present as much peptide sequence as possible to the host immune system. One common means of conjugation method is the maleimide method, which couples the cysteine residue of the peptide to the carrier protein. For peptides with no cysteine residues, one cysteine residue is added to the N- or C-terminus of the peptide so that it may be linked to the carrier protein. As a general guideline, If the peptide originates from N terminal of the protein, coupling to the carrier protein should be at the C-terminus, conversely, the carrier protein is be added to the N-terminus of the peptide. And if the peptide comes for internal of the protein, the carrier protein is conjugated to either N- or C- terminal of peptide.
Here is a summary, KLH is preferred as immunogen for the immunization in many case. When the cost is being considered, BSA or OVA are usually chosen. BSA has high nonspecific binding and ideal for use in immunoassay capture antigens and OVA has been used in immunogens as well as capture antigen.
In addition, in order to generate an immune response or high quality polyclonal antibodies, one of the crucial steps is to design the optimal synthetic peptide antigen. Try OptimumAntigen Design Tool and choose the best antigen. If you have questions about peptide conjugation or synthetic peptide antigen, please contact us at firstname.lastname@example.org
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1. Li, Bowen, et al. "Revealing the immunogenic risk of polymers." Angewandte Chemie International Edition 57.42 (2018): 13873-13876.