Elution of antibodies from affinity columns
Specific signal can be lost
Purified antibodies are not stable
Ideal antibody for affinity purification: Has high affinity for antigen, and can be realeased 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 the affinity to Protein A or Protein G to obtain a total immunoglobulin pool.
Binding to the matrix occures 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. Bound between antibodies and antigen needs to be disrupted to allow the elution.
Elution using extremes pH
- 100 mM glycine pH2.2-2.8
- 100 mM citric acid buffer pH 3.5-4
- 100 mM glycine-NaOH pH 10.5
- 0.05 mM diethylamine pH 11.5
- Sodium borate pH 10
Comments: Elution by extreme pH involves possibility of denaturation of eluted protein as well as protein coupled to the matrix. Precipiation 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 environemtn to cause the release of antigen or antibody and low yields can be obtained. High pH elution can be especially effective for membrane proteins.
Elution at neutral pH:
- Commercially available reagents allow elution at pH around 7.
Comments: Not all antibodies from the pool can be eluted in such conditons and 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 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
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: Elution conditions have to be tested experimentally and established individually for each antigen-antibody pair. In some cases effective elution can not be achived by standard conditions (low pH) and other methods have to employed. However, if using more harsh methods, eluted proteins can be irreversibly denaturated. Elution conditions are some kind of compromise between the achived yield and protein activity.
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 upon the matrix used for coupling efficiency and size of the antigen coupled on the column.