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A description of the different formats in which antibodies are supplied and purification methods in use.
Updated May 11, 2022.
If an antibody is received in an unpurified format, you may need to purify it before using it in your experimental setup. Antibody purification methods range from very crude to highly specific, and the necessary level of purification depends on your intended application for the antibody. Here we briefly overview the most common unpurified antibody formats and antibody purification methods.
Polyclonal antibodies are often available in relatively unpurified forms, such as "serum" or "antiserum". Antiserum refers to the blood serum from an immunized host containing antibodies of interest (as well as other serum proteins and antibodies).
In addition to antibodies recognizing the target antigen, antiserum contains antibodies to various non-target antigens that sometimes react non-specifically in immunological assays. For this reason, raw antiserum is often purified to eliminate serum proteins and enrich the immunoglobulin fraction that specifically reacts with the target antigen.
Monoclonal antibodies can be produced using hybridoma cell cultures (cytokine-secreting cells) and harvested as hybridoma tissue culture supernatants. For further details on how hybridomas are produced, please see the section on monoclonal antibody production.
Ascites fluid is a historical in vivo antibody production method, which is now only used in exceptional cases, ie, when an antibody can't be produced by in vitro technologies.
In this method, monoclonal antibodies are produced by growing hybridoma cells within the peritoneal cavity of a mouse (or a rat). The hybridoma cells are injected into a host's abdomen, where they multiply and generate fluid (ascites), which can be harvested. This ascites fluid contains a high antibody concentration, usually providing higher antibody yields than hybridoma cell culture. However, the ascites fluid also includes many non-specific immunoglobulins from the host.
Unpurified antibody preparations vary significantly in specific antibody concentrations. If the specific antibody concentration of a given unpurified antibody preparation is unknown, one may refer to the following "typical ranges" as a guideline for estimation:
|Tissue culture supernatant
|WB / dot blot
|IHC / ICC
|EIA / ELISA
|FACS / Flow cytometry
Antibody purification is achieved by selective enrichment or specific extraction of antibodies from serum (for polyclonal antibodies), ascites fluid, or cell culture supernatant of a hybridoma cell line (for monoclonal antibodies). Below we describe typical purification methods for polyclonal antiserum or monoclonal ascites fluid/tissue culture supernatant.
Protein A/G purification
Proteins A and Protein G (expressed by Staphylococcus aureus or Streptococcus bacteria, respectively) are antibody binding proteins often used in antibody purification. Protein A/G purification is based on protein A or G's high affinity to the immunoglobulin Fc domain. Protein A/G purification eliminates the bulk of the serum proteins from the raw antiserum. However, it does not eliminate the non-specific immunoglobulin fraction. As a result, the protein A/G purified antiserum may still possess some undesirable cross-reactivity.
Affinity purification isolates a specific protein or group of proteins with similar characteristics using affinity tags. The technique separates proteins based on a reversible interaction between the protein and a specific ligand coupled to a chromatographic matrix.
Antigen affinity purification takes advantage of the specific immunoglobulin fraction's affinity for the immunizing antigen against which it was generated. This purification method eliminates the bulk of the non-specific immunoglobulin fraction while enriching the immunoglobulin fraction that specifically reacts with the target antigen. The resulting affinity-purified immunoglobulin will primarily contain the immunoglobulin of the desired specificity.
Polyclonal antibodies are sometimes pre-adsorbed, meaning they have been adsorbed with other proteins, or serum from various species, to eliminate any antibodies that may cross-react. The solution containing secondary antibodies is passed through a column matrix containing immobilized serum proteins from potentially cross-reactive species. Non-specific secondary antibodies are retained in the column, while highly specific secondaries flow through. The resulting purified antibody should exhibit significantly reduced cross-reactivity.