Techniques of Antibody Purification

This article describes some of the formats in which antibodies are available as well as common purification methods.

Among the widely used techniques for clarification of serum, ascites fluid and the supernatant from tissue culture in the laboratory are centrifugation and filtration, which remove the lipids and particles from the sample, thus preventing the clogging of chromatographic columns.

Buffer exchange with salt removal is required for some samples. Ascites fluid is often used with ammonium sulfate precipitation to bring the immunoglobulins to an adequate concentration.

Antiserum

Polyclonal antibodies are usually prepared in a crude mixture of many specific antibodies to a single antibody, called serum or antiserum. This term refers to blood removed from a host who has been immunized, and from which the coagulation proteins as well as red cells have been removed. The antibodies, also called immunoglobulins, belong to all classes. Other proteins native to serum are also present.

In addition to antibodies specific to the target antigen, there are also antibodies that target other nonspecific antigens. These are responsible for occasional nonspecific binding in immunoassays. Raw serum is therefore often purified for such applications, so that non-immunoglobulin proteins are removed, and immunoglobulins that react specifically with the target antigen are concentrated in the mixture.

Tissue Culture Supernatant

Hybridoma cells may be cultured to produce cytokines, and the supernatant may be harvested. This tissue culture supernatant is rich in monoclonal antibodies.

Ascites Fluid

Another technique to produce monoclonal antibodies is to grow hybridoma cells injected into the mouse (or rat) peritoneal cavity. The multiplication of these hybridoma cells leads to the occurrence of ascites, or fluid accumulation within the abdomen. This fluid is rich in the antibodies secreted by the hybridoma cells, even more than hybridoma tissue culture supernatants.

Unpurified Antibody Concentrations

Preparations of antibodies may have a widely varying range of concentrations of specific antibodies if not purified. If unknown, the concentration of a specific antibody in a particular preparation may be estimated using the following table of typical ranges for reference.

	Tissue culture supernatant	Ascites	Whole antiserum	Purified antibody
WB / dot blot	1/100	1/1000	1/500	1 µg/ml
IHC / ICC	neat–1/10	1/100	1/50–1/100	5 µg/ml
EIA / ELISA	1/1000	1/10000	1/500	0.1 µg/ml
FACS / Flow cytometry	1/100	1/1000	1/500	1 µg/ml
IP	1/10–1/50	1/100	1/50–1/100	1–10 µg/ml
Concentration estimate	1–3 mg/ml	5–10 mg/ml	1–10 mg/ml

Purified Antibodies

One of the following methods is used to purify monoclonal tissue culture supernatant or ascites fluid, as well as polyclonal antiserum:

Protein A/G Purification

The protein A/G purification technique takes advantage of the high binding affinity that the Staphylococcus aureus protein A or Streptococcus protein G has for the Fc domain of the immunoglobulin molecule. It allows most other serum proteins to be eliminated from the raw antiserum, but not any nonspecific immunoglobulin which also binds these proteins. Thus, such purified antiserum may still show some amount of cross-reactivity.

Affinity Purification

The technique of affinity purification allows one protein in particular or a family of proteins to be separated from the antiserum based on the similarity of their reversible interactions with a specific ligand which is bound to a chromatographic matrix.

This method uses the specific affinity that a fraction of the immunoglobulin in the antiserum has for the antigen against which it was raised. This therefore removes most of the nonspecific protein-antibody interactions. The result is an enriched fraction of immunoglobulin, specific to the target antigen with minimal nonspecific binding.

Pre-Adsorption

In some situations pre-adsorption is carried out for polyclonal antibodies. In other words, the antibodies are subjected to adsorption with other proteins or serum, gained from other species, to remove cross-reactive antibody. The result is a highly purified and specific antibody that reacts only with the specific antigen.