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Phage Display of Peptides in Ligand Selection 119
3.6. Characterization of a Phage Peptide as a Mimotope
It is important to characterize the selected phage clones for their ability to
mimic the native antigen. Ideally, two ELISAs should be performed involving
competition of the phage with the native antigen for binding to the parent
antibody and checking the phage does not bind to the constant or framework
regions of the antibody using a relevant isotype control antibody. Examples of
the typical results are shown in Fig. 2B and C. The same ELISA described in
Subheading 3.2 should be used with the following modifications.
3.6.1. Antigen Competition ELISA
Step 3: Various concentrations of antigen competitor (usually 0.1–200 μg/ml)
can be mixed with a constant phage dilution (50 μl of each). The optimal phage
concentration to be used can be determined first by titrating the phage and
taking a dilution at the top of the binding curve where it begins to plateau.
3.6.2. Isotype Control ELISA
Step 1: Wells should be coated with the isotype control antibody and binding
compared to the original antibody.
3.7. Characterization of the Synthetic Peptide
Once it has been established the selected phage clone(s) are true mimotopes,
it is important to prove the peptides are functional in the absence of the phage
framework (data not shown). This can be performed using the ELISA described
in Subheading 3.2 with the following modifications.
Step 3: Various concentrations of peptide (usually 1–500 μg/ml) can be
mixed with a constant phage dilution. The optimal phage concentration to be
used can be determined by titrating the phage and taking a dilution at the top
of the binding curve where it begins to plateau.
3.8. Preparation of the Peptide Affinity Resin (see Note 2)
1) For a 2-ml column weigh out 2 mg of peptide. If the peptide requires organic
solvent (e.g., dimethyl sulfoxide or dimethyl formamide) keep the volume of
solvent to a minimum, approximately 100–200 μl and mix until the peptide is fully
dissolved, then make up to 1 ml with coupling buffer. If the peptide is soluble
dissolve directly into 1 ml of coupling buffer.
2) Mix the NHS-activated Sepharose until an even gel suspension is apparent.
Measure 2 ml of the resin and wash with 15 column volumes (CV) of cold
1 mM HCl.