Epitope affinity chromatography and biophysical studies of monoclonal antibodies and recombinant antibody fragments.
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 is investigated by automated fast-protein liquid chromatography. Purified products are analyzed for purity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. They are then radiolabelled and the immunoreactivity is determined. Ligands are analyzed for secondary structural characteristics by CD and for binding affinity by FQ. Finally, a study is performed to investigate the thermal stability of a recombinant antibody fragment by CD analysis. It is found that simple ligand modifications such as the introduction of a C-terminal cysteine residue can improve purification performance. The FQ studies show that the modified peptide has a higher affinity for antibody. The CD analysis shows that it has a tendency to dimerize because of the formation of disulfide bonds. The versatility of epitope affinity is demonstrated through the purification of a recombinant diabody (dbFv) and by the use of a separate peptide matrix for the purification of an unrelated antibody. All studies result in antibody preparations of high purity and immunoreactivity. The CD analysis of the dbFv shows that it is denatured at 37 degrees C and is 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 plays an important role in the production and characterization of antibody-based reagents for targeted diagnosis and therapy of human diseases.
Murray
A
A
School of Biomedical Sciences, University of Nottingham Medical School, Queen's Medical Centre, UK.
Missailidis
S
S
Price
M R
MR
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000302:isolation & purification*; D002846:Chromatography, Affinity / Q000379:methods*; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes / Q000276:immunology*; D007128:Immunoglobulin Fragments / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000302:isolation & purification
D000911:Antibodies, Monoclonal; D000939:Epitopes; D007128:Immunoglobulin Fragments; D011994:Recombinant Proteins