As you are no doubt aware, carrier proteins play a significant role in antibody production. While immunogenic peptides are generally considered to be good epitopes by themselves, they are too small and lack the additional motifs required to link with class II molecules and T-cell receptors. Thus, they do not have the ability to boost the antibody response.
To solve this problem, immunogenic peptides are coupled with carrier proteins using the appropriate protein cross-linkers. Only when the peptide molecule is attached to a larger carrier protein can it successfully enter the immune pathway and produce the specific antibody capable of attacking the foreign body (i.e., the carrier protein-peptide molecule).
A Little More About Carrier Proteins
Standard peptides only contain about 15 to 20 amino acids. As such, they are too small to elicit the required immune response. This necessitates the need of coupling it to a larger carrier protein. However, since not all carrier proteins are created equally, make sure you choose your carrier protein wisely or you’ll risk jeopardizing the results of your experiment. Here are some simple tips that can help ensure the accuracy of your results.
- Use a larger carrier protein. Larger carrier proteins (MW >60 kDa) have more exposed residues (i.e., functional groups specifically primary amines) available for coupling with peptides. There is also a higher chance that they have the elements required to facilitate T-cell activation.
- Choose one that is genetically distinct from your host. A better immunogenic response can be achieved by using a carrier protein that is genetically distinct from your host.
- Consider solubility. A poorly soluble carrier protein can be difficult to prepare. It may also make the recovery of conjugate more difficult.
Some of the most common carrier proteins include Keyhole Limpet Hemocyanin (KLH), Bovine Serum Albumin (BSA), and cationized BSA.
Keyhole Limpet Hemocyanin (KLH) is considered as the ‘gold standard’ when it comes to carrier proteins. It is also the most widely used carrier protein in antibody production procedures for a number of reasons.
First, KLH is a large protein composed of multiple subunits with MW ranging from 350 to 390 kDa. When these subunits aggregate, they form a KLH molecule with MW ranging from 0.5 to 8 million Daltons.
Second, it contains numerous lysine residues that provide numerous epitopes (primary amines) capable of covalently attaching to haptens, thereby increasing the chance of producing hapten-specific antibodies.
Lastly, since KLH is derived from Megathura crenulata, a gastropod found off the coast of California, it is evolutionarily distinct from mammals. This further strengthens the immune response and reduces the likelihood of getting false positives when working with mammalian sample organisms in immunologically-based research.
Despite the apparent advantages, KLH is far from perfect. Aside from its limited solubility in aqueous solutions, KLH tends to precipitate due to the availability of numerous coupling sites. To circumvent this limitation, stabilized and pre-activated formulations have been formulated and made commercially available.
Bovine Serum Albumin (BSA) may be significantly smaller (MW 67 KDa) than KLH, but it is highly soluble in aqueous solutions and is fully immunogenic as well. Out of its 59 lysine residues, about 30 to 35 are available for coupling. BSA gained popularity in the development of immunoassays, as a blocking agent and molecular weight marker in SDS-PAGE due to the presence of numerous functional groups available for crosslinking, its high solubility and its ready availability.
Cationized BSA (cBSA) is a highly positively-charged protein resulting from the addition of excess ethylene diamine to native BSA. Replacing the negatively-charged carboxyl groups with positively-charged aminoethyl-amide groups increases the number of primary amines available for crosslinking with antigen molecules and increases the immunogenicity of native BSA. It also makes the immune response last for longer periods of time.
