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Phage Display of Peptides in Ligand Selection 123
Further ELISA tests can be performed to analyze the efficiency of the peptide
affinity resin. Characterization of the serum prior to purification in Fig. 3B
illustrates binding of the serum antibodies to the original antigen and two
other antigens, whereas after purification (see Fig. 3C), the eluted antibodies
should show higher relative binding to the original antigen than to the two
other antigens. This indicates enrichment for antibodies binding to the original
antigen via affinity purification using the peptide mimotope. In addition, a
competition ELISA could be performed using the resulting peptide-purified
antibodies (see Fig. 3D). These antibodies should compete with the peptide they
were purified against, but should not compete with other non-specific peptides.
Furthermore, the peptide-purified antibodies should compete with the original
antigen as they share specificity with the peptide mimotope (see Fig. 3D).
Refer to Subheading 3.2 for ELISA protocol, Subheading 3.6 for competition
ELISA and the modifications described earlier in this section.
4. Notes
1. Tips for handling bacteriophage: Bacteriophage should be treated with care, and
the following points should be considered to prevent possible contamination.
a. It is recommended whenever using phage to use filter tips.
b. All work surfaces should be cleaned with 2% bleach prior to and after working
with phage.
c. Pipettes should be cleaned regularly with 2% bleach, certain parts can be
autoclaved (check with the manufacturer).
d. When performing ELISA washes, use a separate piece of paper towel for
blotting. The towel should be placed in a biohazard bag and autoclaved.
e. Autoclave all bacteriophage waste.
2. Peptide affinity ligands: This system can be applied to any peptide selected against
any potential monoclonal antibodies or polyclonal antibodies.
a. The solubility and stability of the peptide will affect the stability of the affinity
column and the number of times the column can be used successfully.
b. This purification system may result in low yields of protein mainly because
antibodies to a single epitope are being selected.
3. Maintenance of the peptide column:
a. For long-term storage, the peptide affinity column should be stored in 20%
ethanol.
b. For sanitation and removal of bacterial contaminants, wash the column with
0.1 M NaOH in 20% ethanol allowing contact for 1 h.
c. To prevent clogging of the column, 0.2 μm, filter all buffers and sample prior
to loading.