Technical information > Protein purification using antibodies

Aim

Needed

• Activated solid support - matrix
• Antibody preparation, polyclonal or monoclonal (more homogeneous)
• Mild and irreversible coupling method, which will retain biological activity of antibodies and allow their proper orientation

 Antibody coupling methods

• Method 1: Random direct coupling via lysine amino groups with multiple attachments
  Note: In case of random coupling, the antibody can be coupled via any available free lysine residue including antigen binding domain and, in that way, decrease the amount of antibody which retain enough biological activity to bind antigen. Therefore, the final binding capacity might be much lower than what is expected. This might be of minor importance when coupling antibodies through free amino groups on the matrix with a spacer arm. In this case antibodies might still be capable of binding small ligands (antigens).

• Method 2: Coupling with orientation via carbohydrate sidechains
  Note: Cannot be used in case of recombinant scFv antibody fragments expressed in E. coli, since they are not glycosylated. Cannot be used if only the Fab' fragments of the antibody are available. Some monoclonal antibodies might have carbohydrate sidechains present in the antigen binding sites.

• Method 3: Coupling with orientation via free hinge region -SH groups in partially reduced Fab' or half-molecule fragments
  Note: Involves some extra steps to obtain reduced antibody fragments.

• Method 4: Indirect coupling and orientation via Protein A or G with cross linking
  Note: This indirect coupling method involves some extra steps, but allows free access to the antigen binding domain. Binding via the antigen binding domain might also occur, but with very low frequency. Protein A or G is used for purification of the total immunoglobulin fraction. Check the specificity of the matrix for different immunoglobulin classes before the experiment. Important note: if planning to purify antigen from a serum sample on such a column, consider that antibodies present in the sample will bind to free sites still available on Protein A or G.
  

Possible limitations

• Amount of antibody bound on the affinity column. Are affinity purified antibodies used, total immunoglobulin fraction (contains from 0.5-10% of antigen-specific antibodies) or monoclonal antibodies?
• Low binding capacity.
• Harsh elution conditions from the column, optimization necessary.
• Limited specificity depending upon the quality of the antibodies.
• Antigen binding affinity will vary between different antibodies, therefore binding and elution conditions must be optimized for every specific case.
  

Recommended literature

• Using Antibodies: A Laboratory Manual, E. Harlow and D. Lane, 1999,
  ISBN: 0879695447; Publisher: Cold Spring Harbor Laboratory Press.
• "Monoclonal antibodies - practical approach" by P. Shepherd and C. Dean, 2000,
  ISBN0-19-963723-7; Publisher Oxford University Press
• "Monoclonal antibodies: principles and practice" by James W. Goding, 1996,
  ISBN 0-12-287023-9; Publisher: Academic Press.