Peptide-Based Drug Design

Lipopeptides 255
Representative results of HPLC chromatograms and mass spectra that we have
obtained are shown in Fig. 1. Also shown are schematic representations of the
peptide and lipopeptide structures.
3.4. Evaluation of Vaccine Efficacy
There are many techniques that can be employed to assess the immunogenicity
of a vaccine candidate, and laboratories interested in pursuing this
objective should also explore other assays suited to their purposes. In this
model, vaccine immunogenicity is measured by determining levels of anti-
LHRH antibodies induced in mice immunized with lipopeptide or a nonlipidated
peptide control. It is important to correlate vaccine immunogenicity with
biological function, and we measure the reproductively capability of vaccinated
female mice as an indication of vaccine efficacy. Techniques that determine
testosterone and oestrogen levels are also useful.
3.4.1. Immunization of Mice
1. Dissolve peptide and lipopeptide vaccines in saline to a concentration of 200
nmol/mL. If necessary, encourage dissolution by warming in a water bath and/or
by sonication. Do not mix by pipet if the inoculant is not soluble; this can result in
insoluble peptide or lipopeptide being trapped in the pipet tip.
2. Immunise 6- to 8-week-old female BALB/c or C57BL6 mice either intranasally
or subcutaneously in the base of the tail with 20 nmol (100 �L) of lipopeptide or
nonlipidated peptide in saline per dose.
3. Perform booster inoculations using the same amount of peptide and lipopeptide 4
wk later (see Note 5).
4. Obtain blood 2 wk following the second dose of vaccine (see Note 6).
5. Prepare sera by leaving blood overnight at 4 ◦ C to coagulate.
6. Centrifuge tubes containing coagulated blood to separate sera.
7. Collect sera and store at −20 ◦ C or use immediately.
3.4.2. Enzyme-Linked Immunosorbent Assay (ELISA)
The working principle of the ELISA described here involves the use of
LHRH peptide-coated wells to capture any anti-LHRH antibodies present in
the sera of vaccinated mice. Captured antibodies are detected through the use
of a horseradish peroxidase-conjugated antibody that is specific for mouse
immunoglobulins and a substrate is then added to induce a color change. In
this case, the substrate utilized is 2,2´-azino-bis 3-ethylbenzthiazoline-sulfonic
acid (ABTS; Sigma-Aldrich, USA), which is converted to a green soluble end
product by horseradish peroxidase (see Note 7). The level of substrate-induced