Antibody purification

Antibody purification is advisable for certain applications, e.g. immunoprecipitation, immunohistochemistry and antibody labeling. Agrisera offers antibody purification of a range of different immunoglobulins:

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

Please note that:

• The total immunoglobulin pool will contain only 0.5-10 % of antigen-specific antibodies. The remaining antibodies are active against other antigens.
• If certain antigen-binding antibodies are lost or an enrichment of non-specific antibodies occurs during the purification process, the signal-to-noise ratio can deteriorate rather than improve.

Affinity purification

To obtain antigen-specific antibodies (from serum or IgY fraction), the following steps are performed:

• Coupling of peptide or customer-supplied antigen to matrix. UltraLink® is used for peptides and Hi trap NHS for proteins. The affinity column can be reused and will be delivered to the customer, together with the purified antibodies.
• Antigen-specific purification of 15-20 ml serum or up to 400 mg of total IgY (4 eggs).
• Specific activity of affinity-purified antibodies confirmed by ELISA.

Theoretical yield from affinity purification is around 0.5-5 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.

When necessary, antibodies will be carefully eluted in two different steps, including elution at neutral pH, to prevent irreversible denaturation of purified antibodies.

To obtain total immunoglobulin fraction, the following steps are performed:

• Isolation of total IgG from 20 ml serum (rabbit, goat, mouse and rat) using Protein G Sepharose 4 Fast Flow.
• Specific activity of affinity-purified antibodies confirmed by ELISA.

In case of very low amounts of protein are available for affinity purification, it can be done using this approach.

For additional information or ordering, please contact us at [email protected].