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Phage Display of Peptides in Ligand Selection 117
A
Absorbance 490nm
1.4
1.2
1
0.8
0.6
0.4
0.2
0
B
1.2
R0 R1 R2 R3 R4 control
phage
mAb
no mAb
no phage
Absorbance 490nm
C
1
0.8
0.6
0.4
0.2
0
1
mAb Isotype control no mA b
Absorbance 490nm
0.8
0.6
0.4
0.2
0
0 0.1 1 2 5 10 50
Antigen (µg / ml)
Fig. 2. Selection and characterization of phage clones as mimotopes. (A) Reactivities
of selected phages from each round (R) of panning on a monoclonal antibody (mAb)
detected by ELISA. (B) Binding of a selected phage clone to the mAb but not to an
antibody of the same isotype shown by ELISA. (C) Recombinant antigen is shown to
compete with the phage clone for binding to the parent mAb by ELISA.
5) Apply anti-M13-horseradish peroxidase (HRP) conjugate (Pharmacia Amersham)
diluted in PBST at 1/5000, apply 100 μl/well and incubate with shaking for 1 h
at room temperature.