Peptide-carrier conjugation

S. Muller

2.1 Introduction

Most authors agree that in order to produce antibodies against small peptides of molecular ratio (ΜΓ) 700–1500, it is necessary to enhance their immunogenicity by coupling them to protein carriers (see Butler and Beiser, 1973). Furthermore, when short peptides are used as the immobilized antigen in solid-phase immunoassays, it is either necessary to modify the peptide (Loomans et al., 1997), or use peptide-carrier conjugates, since peptides of 6–15 residues generally do not bind satisfactorily to plastic surfaces.

The different coupling procedures that have been used to prepare peptide-carrier conjugates are listed in Table 2.1. In many instances, the chemistry of these conjugation reactions has not yet been elucidated. The aim of this chapter is to describe the different techniques in such a way that the investigator is able to choose the most appropriate procedure for his particular peptide. The choice of conjugation procedure is important since the antigenic activity of a peptide may be drastically affected by different coupling procedures (Briand et al., 1992b). The coupling of small molecules to carriers has many applications outside immunology (Pique et al., 1978; Talamo et al., 1968; Widder and Green, 1985). Several comprehensive reviews dealing with chemical modifications of proteins can be consulted to obtain information on the types of chemical intermolecular bridges that can be introduced into proteins (Atassi, 1977a; Boersma et al., 1993; Erlanger, 1980; Feeney et al., 1982; Glazer et al., 1975; Han et al., 1984; Hermanson, 1996; Kiefer, 1979; Means and Feeney, 1995; Peters and Richards, 1977; Rongen et al., 1997; Thorell and Larson, 1978).

Most procedures for preparing peptide-protein conjugates are based on the use of symmetrical or asymmetrical bifunctional reagents which either become incorporated into the final conjugate or activate certain reactive sites of the carrier protein molecule for subsequent linkage with the peptide. During the coupling reaction inter- but also intramolecular bridging can take place. Several factors such as protein concentration, ratio of coupling agent to protein, ionic strength and pH govern the kind of linkage obtained in the final product. For example, at low concentration of reactants, unwanted intramolecular linking reactions often predominate.

Although a wide range of coupling reagents has been reported in the literature, nearly all of them react primarily with the ε-amino group of lysine and the nucleophilic thiol of cysteine and only secondarily with the imidazole group of histidine and the phenolic hydroxyl group of tyrosine (Tables 2.1, 2.2). Only a few compounds such as bisimido esters are group-specific protein reagents.

2.2 Choice of carrier

Carrier molecules are chosen on the basis of criteria such as availability of reactive sites, size, solubility, immunogenicity, commercial availability and cost. The most commonly used carriers are listed in Table 2.3. One of the most important parameters that determines the suitability of a particular carrier molecule is the solubility of the final conjugate, since this may influence the accessibility of antigenic sites. When using the same coupling agent and peptide, some carriers may give insoluble conjugates while others give rise to soluble conjugates. Certain carriers are preferable when the peptide-carrier ratio is to be determined by amino acid analysis. Keyhole limpet haemocyanin (KLH), for instance, is not appropriate in this case since it has a high molecular weight and commercial preparations usually contain many impurities. In our laboratory, we use bovine serum albumin (BSA) for preparing peptide conjugates intended for use as antigens, and ovalbumin for preparing conjugates to be used for immunization. When this combination of carriers is used, BSA can be included as blocking agent in immunoassays without interference by the antiovalbumin antibodies present in the antipeptide-carrier antiserum.

Apart from presenting the peptide in a polyvalent fashion, the carrier can also exert an effect on the structure of the associated peptide and hence on its immunogenicity and antigenicity (Bahraoui et al., 1986b; Friede et al., 1994; Mariani et al., 1987). According to the point of attachment of the peptide to the carrier and the electrostatic and hydrophobic environment of this site, peptides can adopt different structures. In this regard, the use of liposomes as carrier of peptides presents several advantages over the protein constructs in that the surface of the liposome being uniform, it is probable that carrier effects on the structure of the peptide will be uniform (Friede et al., 1993c, 1994).

2.3...