DFE-Based Affinity Chromatography: A Technique for Purification of 2F5 Monoclonal Antibodies from a Crude Sample Using High Ionic Strength-Based Elution Conditions
Transcrição
The DFE or DsRed-2F5-Epitope is a recombinant fusion protein comprising DsRed, a fluorescent protein, and the 2F5 epitope – a segment of the HIV-1 envelope glycoprotein antigen. This epitope has a high affinity for its paratope – a region in the complementarity-determining region on the 2F5 monoclonal antibody.
To purify 2F5 monoclonal antibodies from a crude sample, take a column packed with porous resin beads containing immobilized DFE affinity ligands on their surface. Rinse the column with a suitable equilibration buffer to maintain the pH and prevent non-specific interactions between the sample components and the resin in the subsequent steps.
Load the sample containing the target 2F5 monoclonal antibodies into the column. Gradually, the antibodies in the sample begin to flow through the column or the stationary phase.
During the flow, the antibodies interact reversibly with the DFE affinity ligands immobilized on the stationary phase. In these interactions, the paratope on the 2F5 antibody binds specifically to its complementary epitope on the DFE ligand.
Wash the column with an equilibration buffer to remove undesirable sample bound non-specifically to the column. Pass an elution buffer through the column.
The buffer's high ionic strength destabilizes the interactions between the DFE ligands and the 2F5 monoclonal antibodies. Consequently, the monoclonal antibodies elute as fractions from the column.
Collect the purified antibody fractions, and store them for downstream applications.
