Conjugation of Haptens Peptides to Carrier Proteins

Low-molar mass molecules, such as, for example, chemically synthesized peptides, are in most cases not able to induce antibodies in animals therefore, these molecules have to be covalently coupled 

To get a directed coupling of a peptide via its N-terminal NH2-group, the introduction of a monochloroacetylglycyl residue during peptide synthesis is recommended. Of course, coupling via NH2-, SH-, or carboxyl groups of peptide side chains is also possible. 

Production of antiserum with high titer and specility is done by trial and error, especially because each immunized animal gives antisera with different characteristics therefore, several groups of animals should be immunized with different antigen preparations. 

Universal haptens, suitable especially in immunohistochem-istry, dot blots. Western blots, or other kinds of immunoassays, are, for instance, biotin, fluorescein, or digoxigenin. 

Some coupling reagents suitable for preparation of hapten-carrier conjugates are given in Table 4.1. 

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Figure 19.13 Conjugation to KLH often can cause precipitation due to the high-molecular weight of the carrier protein. The conjugation of [Met5]-enkephalin to KLH yields a soluble immunogen if the level of EDC addition is about 0.1 times that typically used with BSA as a carrier. This figure shows the gel filtration pattern on Sephadex G-25 after completion of the crosslinking reaction. The first peak is KLH and the second peak is excess peptide. Depletion of the peptide peak correlates to hapten-carrier conjugation.

This chapter describes the design, preparation, and use of hapten-carrier conjugates used to elicit an immune response toward a coupled hapten. The chemical reactions discussed for these conjugations are useful for coupling peptides, proteins, carbohydrates, oligonucleotides, and other small organic molecules to various carrier macromolecules. The resultant conjugates are important in antibody production, immune response research, and in the creation of vaccines. 

Synthetic haptens mimicking some critical epitopic structures on larger macromolecules are often conjugated to carriers to create an immune response to the larger parent molecule. For instance, short peptide segments can be synthesized from the known sequence of a viral coat protein and coupled to a carrier to induce immunogenicity toward the native virus. This type of synthetic approach to immunogen production has become the basis of much of the current research into the creation of vaccines. 

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