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Centrifugation and filtration are standard laboratory techniques for sample clarification of serum, ascites fluid and tissue culture supernatant. These techniques remove lipid and particle matter that can block chromatographic columns.
For some materials, buffer exchange and desalting may also be necessary. Ammonium sulfate precipitation is a further preparation step often used with ascites fluid to concentrate the immunoglobulins.
Polyclonal antibodies are often available in relatively unpurified forms, described as "serum" or "antiserum". Antiserum refers to the blood from an immunized host from which clotting proteins and red blood cells have been removed. The antiserum will contain antibodies/immunoglobulins of all classes as well as other serum proteins.
In addition to antibodies that recognize the target antigen, the antiserum also contains antibodies to various non-target antigens that can sometimes react non-specifically in immunological assays. For this reason, raw antiserum is often purified, to eliminate serum proteins and to enrich the fraction of immunoglobulin that specifically reacts with the target antigen.
Monoclonal antibodies may be grown as hybridoma cell cultures (cells secreting cytokines) and harvested as hybridoma tissue culture supernatants. For further details on how hybridomas are produced, please see section on monoclonal antibody production.
Monoclonal antibodies can be produced by growing hybridoma cells within the peritoneal cavity of a mouse (or rat). When injected into a mouse, the hybridoma cells multiply and produce fluid (ascites) in its abdomen. This fluid contains a high concentration of antibody which can be harvested. This usually provides higher antibody yields than hybridoma cell culture.
Unpurified antibody preparations vary significantly in specific antibody concentration. 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||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|
|Concentration estimate||1–3 mg/ml||5–10 mg/ml||1–10 mg/ml|
Polyclonal antiserum or monoclonal ascites fluid/tissue culture supernatant is commonly purified by one of two methods:
Protein A/G purification
Protein A/G purification uses the high affinity of Staphylococcus aureus protein A or Streptococcus protein G 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 a small amount of undesirable cross reactivity.
Affinity purification isolates a specific protein or group of proteins with similar characteristics. The technique separates proteins on the basis of a reversible interaction between the proteins and a specific ligand coupled to a chromatographic matrix.
Antigen affinity purification takes advantage of the affinity of the specific immunoglobulin fraction for the immunizing antigen against which it was generated. Antigen affinity purification results in the elimination of the bulk of the non-specific immunoglobulin fraction, while enriching the fraction of immunoglobulin that specifically reacts with the target antigen. The resulting affinity purified immunoglobulin will conaitn primarily the immunoglobulin of desired specificity.
Polyclonal antibodies are sometimes pre-adsorbed. This means they have been adsorbed with other proteins, or serum from various species, etc., to eliminate any antibody that may cross-react. The resulting purified antibody should be very pure and specific and any cross reactivity should be significantly reduced.