Immunogen synthesis

Success in the synthesis of new catalytic antibodies (CAs) depends on the efficiency of each of the following steps 1) hapten design, 2) immunogen synthesis, 3) preparation of the enzymatic tracer 4) generation and purification of antibodies and 5) kinetic assays. 

Development of conjugate and peptide vaccines requires the typical organic synthesis process and purification. This is a new area for vaccine technologists. Again, the main concern is to maintain the immunogenicity of the vaccine candidate during the chemical reaction and purification steps. 

Table 1 Guidelines for the design and synthesis of an immunogen hapten...

Berkman C., Underiner G., Cahsman J. Synthesis of an immunogenic template for the generation of catalytic antibodies for (-) cocaine hydrolysis. J. Org. Chem. 61 5686, 1996. 

Selkirk, M.E., Nielsen, L., Kelly, C., Partono, F., Sayers, G. and Maizels, R.M. (1989) Identification, synthesis and immunogenicity of cuticular collagens from the filarial nematodes Brugia malayi and Brugia pahangi. Mokcular and Biochemical Parasitology 32, 229—246. 

The exact mechanism of the immunogenicity of species-specific rDNA proteins is unknown, but is believed to be attributable to (1) the addition of extra amino acid residues during synthesis, which the host reads as foreign (2) denaturing of the native molecules or (3) contamination by E. coli polypeptides or lipopolysacchar-ides. 

Scheme 1.7 Solid-phase synthesis of an immunogenic heptasaccharide by van Boom et al.

Synthesis of Immunogen Morphine is first converted to 3-o-carboxymethyl-morphine by reacting the free base with sodium-p-chloroacetate in absolute ethanol ... 

Synthesis of Immunogen Chlordiazepoxide as suspension inN-methylformamide is treated with HC1 in dioxane to yield a pale-yellow solution. The resulting mixture is cooled to -30 °C and isoamyl nitrite in dioxane is added. The solution is stirred at - 30° to - 40 °C and aqueous ammonium sulfamate is added with continuous stirring. 

Synthesis of Immunogen (Hapten) The barbiturate, 5-ally 1-5-(1-carboxyisopropyl) barbituric acid (I) is first converted to 5-allyl-5-(1 -p-n i trophenyloxycarbonylisopropyl) barbituric acid (II) by the interaction of the base with /7-nitrophenol in N, N-dimethylformamide (DMF) as shown below ... 

Peptides have many desirable properties as components of synthetic vectors. Peptide synthetic chemistry is well established, with the convenience of automated synthesis resulting in a well-defined, high-purity product of low toxicity and immunogenicity for in vivo use. Furthermore, even short peptides of 7 to 30 amino acids can accommodate enormous structural diversity, functionality, and combinations of properties. 

The latter approach has been followed in our laboratory. For instance, in studies related to the synthesis of potent multivalent sialoside inhibitors of influenza virus hemagglutinin [34], it became of interest to evaluate the immunogenicity of neoglycoproteins containing sialosides as sole immunodominant hapten. To this end, acetochloroneuraminic acid (1) was transformed into allyl glycoside 2 (Scheme 1) which upon reductive ozonolysis afforded aldehyde 3 in excellent overall yields [35], Reductive amination of 3 to bovine serum albumin (BSA) and tetanus toxoid provided immunogenic vaccines from which specific rabbit IgG anti-sialic acid antibodies were obtained [36]. In order... 

Fig. 28. Synthesis of labeled DNA probes. A Labeled DNA can be generated using different enzymes (Klenow fragment of DNA polymerase or a terminal transferase) to incorporate labeled nucleotides into specific DNA sequences. Probes can be labeled using radioactive nucleotides or nucleotides labeled with an immunogenic molecule such as biotin. B The labeled probe is then hybridized to the target nucleic acid, which is either bound to a membrane or in a tissue section or cell. An antibody is then used to detect the non-radioactively-labeled probe. C The antibody may be conjugated to a fluorescent or chemiluminescent dye, or an enzyme that produces a color reaction. The target nucleic acid is thus visualized.

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