Technical information > Elution of antibodies from affinity columns

Limitations

• Specific signal can be lost
• Purified antibodies are not stable
  
  An ideal antibody for affinity purification has high affinity for antigen, and can be released from the antigen by a gentle change in environment (pH). Ease of disruption of an antigen/antibody complex is not related to affinity (Goding 1996). Therefore, both high and low affinity antibodies require the same elution conditions.
  
  

Elution of antibodies

1. Antibodies are purified based on their affinity to Protein A or Protein G to obtain a total immunoglobulin pool. Binding to the matrix occurs mainly via Fc part of the antibody.

• Elution using 100 mM glycine pH 2.5-2-7
• Elution in neutral pH, using some of commercially available reagents

2. Either antigen or antibody is immobilized on the appropriate matrix. The bond between antibody and antigen needs to be disrupted to allow the elution.

• Elution using extreme pH
  • Low pH
    • 100 mM glycine pH2.2-2.8
    • 100 mM citric acid buffer pH 3.5-4
  • High pH
    • 100 mM glycine-NaOH pH 10.5
    • 0.05 mM diethylamine pH 11.5
    • 10-50 mM Sodium borate pH 10
  • Comments: Elution by extreme pH involves the possibility of denaturation of the eluted protein, as well as the protein coupled to the matrix. Precipitation problems can occur as well, therefore immediate neutralization is recommended (using 1 M Tris pH 9 or other buffers). Extreme pH might not give enough change in the environment to cause the release of antigen or antibody, leading to low yields. High pH elution can be especially effective for membrane proteins.
• Elution at neutral pH
  • Commercially available reagents allow elution at a pH of around 7.
  • Comments: Not all antibodies from the pool can be eluted in such conditions and an additional elution step is required.
• Elution of antibodies directly from Ni-NTA columns
• Elution using chaotropic ions
  • 3 M KSCN or 3.5 M NaSCN
  • Comments: SCN ions will absorb at 280 nm and interfere with UV monitoring of the elution process. There is a risk that the eluted antibody/antigen is not going to be active.
• Elution using denaturants (Last instance to try)
  • 6-8 M urea or 3-4 M guanidine-HCl
    • Comments: There is a risk for irreversible denaturation of the eluted protein. More information can be found in "Using Antibodies: A Laboratory Manual" (Harlow and Lane, second edition, September 2013).

General comments

Theoretical yields of affinity purifications

• 5 mg of peptide coupled on affinity column: from 10-25 mg of specific antibodies.
• 1 mg of protein coupled on affinity column: from 0.5-2 mg of specific antibodies.
  
  Note that total yields will depend on the matrix used for coupling efficiency, as well as the size of the antigen coupled on the column.