Conjugation of Peptides to Carrier Proteins

In general, short peptides are poor immunogens, so it is necessary to conjugate them covalently to immunogenic carrier proteins to raise effective anti-peptide antibodies. These carrier proteins provide necessary major histocompatibility complex class II/T-cell receptor epitopes while the peptides can then serve as B-cell determinants. Keyhole limpet hemocyanin (KLH) and thyroglobu-lin are examples of carriers that are commonly used to generate polyclonal anti-peptide antibodies. We generally avoid using bovine serum albumin because the high levels of anti-bovine serum albumin antibody generated can interfere with subsequent studies on tissue culture cells grown in media containing bovine sera.

The peptides are covalently conjugated to the carrier molecule using an appropriate bifunctional reagent—the most straightforward coupling methodologies involve the amine or sulfhydryl groups of the peptide. Substantial antibody titres are also usually generated against determinants present on the carrier molecules. In general, such anticarrier antibodies present few problems in polyclonal antipeptide antibodies and may anyway, be adsorbed out on a matrix of carrier bound to agarose. When making monoclonal antibodies, however, the substantial anticarrier response may mask the frequently weaker anti-peptide response, resulting in few peptide-spe-cific hybridomas being isolated. A variety of alternative approaches to the use of conventional peptide-carrier conjugates have been developed including, for example, the multiple antigenic peptide (MAP) system (9). The MAP system makes use of the epsilon-amino group of lysine residues to generate a branched core matrix that can be used as a scaffold for subsequent peptide synthesis. This system can be employed to deliver high densities of single, defined peptide antigens or to generate B-cell and T-cell epitopes attached to the same MAP scaffold (10). Nevertheless, MAP synthesis products can be difficult to analyze by high-performance liquid chroma-tography and mass spectrometry because of their large mass.

The most straightforward carrier-peptide conjugation procedure uses glutaraldehyde as the bifunctional reagent, which crosslinks amino groups on both carrier and peptide. In our experience, glut-araldehyde conjugation is reliable, easy and effective, and generates good antipeptide antibodies even with short peptides. m-Maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) can be used to crosslink the thiol group of cysteine on the peptide to an amino group on the carrier. The MBS method generates a somewhat better defined conjugate, but it involves a slightly more involved procedure and requires the presence of a reduced cysteine residue at one end of the peptide (this is frequently added to the sequence during synthesis specifically for conjugation purposes). If the chosen peptide sequence contains an internal cysteine residue, coupling via MBS should be avoided.

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