Antibody purification

Antibody purification is advisable for certain applications as for example: immunoprecipitation, immunohistochemistry, antibody labelling. AgriSera has the experience and commitment to advise on and perform the purification of a range of different immunoglobulins:

Rabbit, goat and mouse IgG
Hen IgY
Rabbit and mouse IgM

Theoretical yield of affinity purification is around 5-10 mg of antigen-specific antibodies per 1 mg of peptide coupled to the support or
0.5-2 mg of antigen-specific antibodies per 1 mg of protein coupled to the support.

Are there any disadvantages of affinity purification?
Yes, pools of antibodies can be difficult to elute from the affinity column and may therefore be lost during the purification process. Note that antibodies which are most difficult to elute will not be those with highest affinity, what is commonly thought.

According to Goding highest affinity interactions are not the ones that require the strongest denaturing conditions to disrupt. Affinity and the ease of disruption of the antibody antigen bound are not necessarily related.
For example, particular antibody can have very high-affinity binding which is critically based on the ionization of a histidine residue. Therefore slight changes of pH could cause drastic loss of this affinity by modifying the charge on this residue.

What are the advantages of affinity purification?

Isolation of antigen-specific antibodies; note that the total immunoglobulin pool will contain only 0.5-10 % of antigen-specific antibodies. The remaining antibodies are active against other antigens.
Purification format - immunoglobulins can be purified to give a total (IgG or IgY) fraction or be subjected to a process of affinity purification utilising the antigen (protein or peptide) coupled to an appropriate support.

IgY purification

Standard purification includes: purification of IgY from 3 pre immune and 6 immune eggs
Yield: 300 - 500 mg of around 80 % pure IgY
Scale up purification from larger amount of eggs (12 and more) - Please, inquire

Affinity purification

To obtain total immunoglobulin fraction (using Protein G)

AgriSera offers purification of:
- Up to 25 ml serum
- Up to 50 ml serum
- Up to 100 ml serum
- More than 100 ml serum
To obtain antigen-specific antibodies (from serum or IgY fraction)
- Example: Coupling of the antigen: protein (>1 mg) or peptide (5 mg and more) to the suitable support

Yield: Few milligrams of antigen specific antibodies

Safety: AgriSera will also confirm by ELISA specific activity of purified antibodies.

Antigen column: supplied to the customer after purification

Alternative service: Preparation of a peptide or protein column only. Please, inquire.

Provide a peptide (min. 5 mg ) or protein (min. 0,5 mg) and in return you will receive a column ready for running affinity purifications of your serum samples together with a simple protocol.

Please, inquire

Important note:
Where necessary, antibodies will be carefully eluted in two different steps, including elution at neutral pH, to prevent irreversible denaturation of purified antibodies. Check also: Antibody elution

When antibodies are purified AgriSera can give assistance during further antibody characterisation using a range of techniques such as Western blotting, ELISA, immunohistochemistry and immunoprecipitation.

Some procols for antibody purification can be found in: Using Antibodies: A Laboratory Manual, E.Harlow and D.Lane, 1999,
ISBN: 0879695447; Publisher: Cold Spring Harbor Laboratory Press.

Last changed: 2009-04-02 by WebMaster