This article discusses the purification of 2F5 monoclonal antibodies using a DFE affinity column. The process involves specific interactions between the antibodies and the DFE ligands, allowing for effective separation and collection of the target antibodies.
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.
First, prepare 100 milliliters of either the clarified plant extract containing 2F5 or the supernatant from the preferred cell-based expression system, which also contains 2F5. Next, prepare the equilibration buffer and the high-ionic-strength elution buffer, as outlined in the text protocol. Flush the chromatography system with the buffers.
Mount a DFE affinity column on the chromatography system, and equilibrate with 5 CV of equilibration buffer at a flow rate of 1 milliliter per minute. Monitor the UV absorbance at 280 nanometers. Then, load 80 milliliters of either the clarified plant extract or the supernatant onto the column at a flow rate of 0.5 milliliters per minute to guarantee a contact time of 2 minutes. Collect the flow-through samples in 2-milliliter fractions for breakthrough curve reconstruction.
Store the flow-through samples at 4 degrees Celsius, if immediate sample analysis is not possible. After this, wash the column with 6 CVs of equilibration buffer. Collect a sample at the beginning, middle, and end of the wash. Elute the mAb 2F5 with 5 volumes of high-ionic-strength elution buffer. Collect the DFE fraction when the UV signal at 280 nanometers has increased to 5 milliabsorbance units above the baseline.
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