Peptide-Based Drug Design

258 Chua et al.
Table 1
Incidence of Pregnancy Following Inoculation with Peptide
Immunogens
Peptide immunogen Administered in Incidence of pregnancy
TH-Lys-LHRH (in saline) Saline 4 /5
TH-Lys (Ser2Pam2Cys)- LHRH Saline 0 /5
TH-Lys-LHRH (in CFA) CFA 0 /5
Inoculated female mice were housed with male mice at 1:2 or 1:3 ratios of males to
females for a period of 3 wk. The male mice were rotated weekly between each cage so
that each group of female mice was exposed to every male. Following removal of the
males, female mice were observed for litter production over a period of 3 wk.
that litter production by a particular female can be confirmed. If female mice are
housed together, be sure to remove those that have dropped litters along with their
offspring.
Table 1 summarizes the results of a fertility trial using lipidated and nonlipidated
peptides. Fertility is totally inhibited in those groups that received lipidated
peptide or nonlipidated peptide administered in CFA.
4. Notes
1. Piperidine has a strong odor and many prefer the use of the odorless DBU reagent
(35).
2. The cleavage reagent should be prepared fresh.
3. Longer time periods for cleavage may be necessary for some sequences, particularly
those containing multiple arginine residues.
4. In those cases where peptides do not dissolve easily in 0.1% aqueous TFA,
addition of acetonitrile may be necessary.
5. As a positive control, a separate group of mice can be inoculated with the same
amount of the nonlipidated peptide emulsified in CFA and boosted with nonlipidated
peptide emulsified in IFA
6. If required, mice can be bled before the second inoculation to obtain an early
indication of vaccine immunogenicity
7. There are many enzyme–substrate combinations that can be utilized in an ELISA,
and investigators should explore which systems work best for their purposes.
Ideally, they should be stable, safe, inexpensive, and easy to optimize. Commonly
used combinations include p-nitrophenyl phosphate (pNPP), which is converted
to the yellow p-nitrophenol by alkaline phosphatase and o-phenylenediamine and
3,3´5,5´- tetramethylbenzidine base, which is converted by peroxidase to orange
and blue end products.