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Phage Display of Peptides in Ligand Selection 121
gel electrophoresis could be used to analyze each fraction and the wash
fractions; however, there will be no distinction between total serum antibodies
and antibodies that bind to the peptide and have been eluted from the column.
Therefore, we recommend performing an ELISA using the native antigen as
the purified antibodies should bind to the same antigen epitope that the peptide
mimics. An example of this is shown in Fig. 3A. The methods described in
Subheading 3.2 can be used with the following modifications.
Step 1: Coat wells of a microtiter plate with 2–10 μg/ml antigen.
Step 3: The original, wash and eluted fractions should be diluted 1:10–1:50
in PBST.
Step 5: Anti-human IgG conjugated to HRP should be used at the manufacturers
suggested concentration (usually 1/5000 dilution for Chemicon AP113P).
The ELISA results should indicate which eluted fractions should be retained;
these should be pooled and dialyzed into PBS and concentrated using an
A
0.5
Absorbance 450nm
B
Absorbance 450nm
0.4
0.3
0.2
0.1
2
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
prepurificati
on
Flow PBS wash1 Bo rate
wash
PBS wash2 Elution1 Elution2 Elution3 Elution4 Elution5 Elution6
0
1000 10000 100000 1000000
Dilution
Fig. 3. (Continued)
Original antigen
Antigen 1
Antigen 2