Benefits of Immunomagnetic Separation for Epitope Identification in Clinically Important Protein Antigens: A Case Study Using Ovalbumin, Carbonic Anhydrase I and Tau Protein

摘要

Immunomagnetic separation (IMS) with specific antibody as affinity ligand immobilized on a magnetic carrier has several advantages in comparison with standard column separation procedures. Epitope mapping enabling identification and characterization of protein structures reactive with the antibody represents one possible application of IMS. We used epitope extraction technique based on the proteolytic digestion of the target protein followed by capturing of a specific peptide fragments by the antibody immobilized on the solid phase. Magnetic particles coated with antibody molecules were first incubated with the prepared mixture of peptides. After specific binding of peptide fragments comprising the epitope sequences, the beads were washed to remove non-epitope peptides. Captured epitope-peptides were then eluted in small volume of 0.05% TFA. Elution fractions were finally analyzed without any modification by mass spectrometry. In this work the results and experience gained in epitope mapping of three clinically important proteins (ovalbumin, carbonic anhydrase I and tau protein) are discussed.