Home › Immunology › Activated Cross-linked Agarose for the Rapid Development of Affinity Chromatography Resins - Antibody Capture as a Case Study
Immunology JoVE (Open Access) Citable · DOI

Activated Cross-linked Agarose for the Rapid Development of Affinity Chromatography Resins - Antibody Capture as a Case Study

DOI: 10.3791/59933-v
What you'll learn
  • ✓ Couple epitope ligands to activated cross-linked agarose resin
  • ✓ Develop affinity chromatography resins for monoclonal antibody capture
  • ✓ Purify antibodies from plant extracts and cell culture supernatants
  • ✓ Validate resin performance using clarified biological samples
Protocol

In this procedure, a DsRed-based epitope ligand is immobilized to produce a highly selective affinity resin for the capture of monoclonal antibodies from crude plant extracts or cell culture supernatants, as an alternative to Protein A.

Difficulty
advanced
Total time
~4–6 hours (coupling + equilibration + purification run)
Biosafety
BSL-1

Steps

1
Couple DsRed epitope ligand to activated agarose

Immobilize the DFE (DsRed-based epitope ligand) onto the activated cross-linked agarose resin by coupling chemistry. This creates the affinity matrix for selective antibody capture.

▶ 01:05
2
Test monoclonal antibody purification from plant extracts

Apply clarified plant extract or cell culture supernatant to the coupled resin and perform affinity chromatography to capture and elute monoclonal antibodies. Assess purification efficiency as an alternative to Protein A.

▶ 03:43
3
Analyze affinity resin performance and yield

Characterize the developed affinity chromatography resin by evaluating binding capacity, selectivity, and antibody recovery from crude biological samples using the purification results.

▶ 05:34
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