Knowledge
by
experience

Preparation of immunoaffinity columns by antibody coupling

Aim:
Antigen purification or removal from a complex mixture

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.
  • Method 2: Coupling with orientation via carbohydrate side-chains.
  • Method 3: Coupling with orientation via free hinge region -SH groups in partially reduced Fab' or half-molecule fragments.
  • Method 4: Indirect coupling and orientation via Protein A or G with crosslinking.
Important notes:
  • Method 1: In case of random coupling antibody can be coupled via any available free lysine residue, including antigen binding domain and that way decreasing the amount of antibodies which retain biological activity to bind antigen. Therefore final binding capacity might be much lower from 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: Can not be used in case of recombinant scFv antibody fragments expressed in E.coli, since they are not glucosylated. Can not be used if only Fab' fragments of antibody are available. Some monoclonal antibodies might have carbohydrate side-chains present in the antigen binding sites.

  • Method 3: Involves some extra steps to obtain reduced antibody fragments.

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


Possible limitations:
  • Amount of antibodies 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, optimisation necessary.

  • Limited specificity depending upon the quality of antibodies.

  • Antigen binding affinity will vary between different antibodies, therefore binding and elution conditions have to be optimised in every 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.

Last changed: 2009-04-02 by WebMaster