Cell Culture Clarified Supernatant vs. Purified Antibodies
The purification of antibodies involves the specific isolation or selective enrichments of antibodies derived from polyclonal antibodies (serum), cell culture supernatant of monoclonal antibodies (hybridoma cell line), or ascites fluid. The method of purification ranges from highly specific to very crude. Below we cover some of the features of each.
Cell Culture Supernatant
The production of monoclonal antibodies is possible through the use of hybridoma cell cultures (cells that secrete antibodies) and harvesting as hybridoma cell culture supernatants. The most simply obtained source of monoclonal antibodies will consist of cell culture supernatants for the majority of immunochemical processes. These supernatants have a concentration high enough for the majority of assays.
During the collection of supernatants containing antibodies, it is important to allow the cultures to grow until the end of the hybridomas exponential growth phase. This enables the gathering of higher-titer supernatants. Permitting the death of the cells should not impact antibody quality.
The purification of antibodies is accomplished through selectively enriching or specifically extracting antibodies from the cell culture supernatant of a hybridoma cell line for monoclonal antibodies. Below, we cover some of the common purification processes for monoclonal ascites fluid/cell culture supernatant or polyclonal antiserum.
Protein A/G purification
Protein G and Protein A are bacterial proteins that bind antibodies and are commonly used for their purification. The purification of these proteins is based on high affinity to the immunoglobulin Fc domain. The purification of protein A/G removes the greater part of the serum proteins from the raw antiserum. However, the purification does not eradicate the fraction of the non-specific immunoglobulin. Consequently the purified antiserum of protein A/G may still include some unwanted cross-reactivity.
Affinity Tag Purification
This form of purification isolates a particular protein or a collection of proteins through the use of affinity tags. This method uses a reversible interaction between the protein and a chromatographic matrix coupled to a specific ligand to separate the proteins.
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