Murray, A.; Missailidis, S. and Price, M.R.
Epitope Affinity Chromatography and Biophysical Studies of Monoclonal Antibodies and Recombinant Antibody Fragments.
Journal of Chromatographic Science, 40(6) pp. 343–349.
Peptide epitope affinity chromatography is a powerful technique for the purification of antibodies. This study aims to demonstrate the versatility of the technique and to show how biophysical techniques such as Circular Dichroism (CD) and Fluorescence Quenching (FQ) can aid the rational design of affinity ligands and characterization of antibody based reagents.
The performance of a number of peptide ligands for the purification of a range of different antibodies and recombinant fragments was investigated by automated FPLC. Purified products were analyzed for purity by SDS-PAGE. They were then radiolabelled and the immunoreactivity was determined. Ligands were analyzed for secondary structural characteristics by CD and for binding affinity by FQ. Finally, a study was performed to investigate the thermal stability of a recombinant antibody fragment by CD analysis.
It was found that simple ligand modifications such as introduction of a C-terminal cysteine residue can improve purification performance. FQ studies showed that the modified peptide had higher affinity for antibody. CD analysis showed that it had a tendency to dimerise due to the formation of disulphide bonds. The versatility of epitope affinity was demonstrated by purification of a recombinant diabody (dbFv) and by the use of a separate peptide matrix for the purification of an unrelated antibody. All studies resulted in antibody preparations of high purity and immunoreactivity. CD analysis of the dbFv showed that it was denatured at 37°C and was therefore unsuitable as a targeting reagent for use in humans in its present form.
It is concluded that epitope affinity chromatography, coupled with biophysical analyses have an important role to play in the production and characterization of antibody based reagents for targeted diagnosis and therapy of human diseases.
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