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Benefits of immunomagnetic separation for epitope identification in clinically important protein antigens

Authors: Jankovičová Barbora | Svobodová Zuzana | Hromádková Lenka | Kupčík Rudolf | Řípová Daniela | Bílková Zuzana

Year: 2014

Type of publication: ostatní - přednáška nebo poster

Page from-to: nestránkováno

Titles:

Language	Name	Abstract	Keywords
eng	Benefits of immunomagnetic separation for epitope identification in clinically important protein antigens	Immunomagnetic separation (IMS) with specific antibodies as affinity ligand immobilized on magnetic carriers has several advantages in comparison with standard column separation procedures. The special advantages of IMS are the fast and simple handling of a sample using magnetic forces, which makes this type of separation ideal for automated analysis systems usable in wide range of applications in the biosciences. One possible application is epitope mapping, which is used for identification and characterization of protein structures reactive with specific antibodies. We used epitope extraction technique based on the specific proteolytic digestion of a target protein followed by immunoaffinity capturing of a specific peptide fragment by the antibody immobilized on the solid phase. Silica superparamagnetic microparticles developing a mean magnetic moment only in the presence of an external magnetic field were used as solid phase, which enables easy and quick separation of particles from the solution by application of simple block magnet and easy resuspension by removing of the magnet. Magnetic particles coated with antibody molecules were incubated with a peptide mixture, after specific binding of peptide fragments comprising epitope sequences beads were washed to remove non-epitope peptides and captured epitope-peptides were subsequently eluted in small volumes of 0.05% TFA. Elution fractions were analyzed 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.	immunomagnetic separation; superparamagnetic beads; epitope mapping; epitope extraction; ovalbumin; carbonic anhydrase I; tau protein