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Posted on Wed, Jul 24, 2019

Antibody Purification

Antibodies are an important part of the immune system. When the body is infected with an antigen, the immune system generates an antibody specific to that antigen. The techniques that are routinely used in biotechnology capitalizes upon this natural immune process. Antibodies are used in many research applications as well as in immunoassays for disease detection. We use the specificity of the antigen/antibody binding for immunoprecipitation and ELISA assays. We use flurophore-conjugated or enzyme-tagged antibodies for labeling molecular targets on individual cells and whole tissue. We use antibody purification to obtain antibodies for biosensors to detect disease. These antibodies, depending on the application, are commonly obtained by antibody purification from humans, rats, rabbits, mice, and chicken. 

Free PDF guide:  "Basic Guide to Recombinant Protein Purification" 

Polyclonal vs Monoclonal antibodies

The antibodies that we use are obtained from serum (polyclonal antibodies), ascites fluid, or from a hybridoma cell line (monoclonal antibodies). Polyclonal antibodies are collected from different B cells, which means there is more variety in antigen epitope recognition. Monoclonal antibodies to a specific antigen are identical and all recognize the same epitope. The type of antibody desired depends on the goals of the procedure at hand.

 

Three main methods for antibody purification

For antibody purification it is essential to match the purification technique with the type of antibody and intended purpose. There are three main methods for antibody purification. The most general and least specific method is to simply separate antibodies from other proteins based on size, structure, and general chemistry. This method results in the purification of all antibodies regardless of type and antigen-binding. The next step up in specificity is to separate a particular class of antibodies (e.g. IgG, IgM, IgA, IgL, IgE.) This is accomplished by using proteins such as protein A or G, which have affinities to particular classes of antibodies. The most specific method of antibody purification is antigen-specific capture. This method often requires that the antigen be bound to a solid support. Magnetic beads are particularly useful for antigen-specific and class-specific antibody purification. More traditionally, resin columns or beads have been used as a solid support. Once the antibody is bound to the antigen or capture protein it must be collected. Antibody binding is most effective at physiological pH. If the pH is increased or decreased to an extreme it will disrupt the binding between antibody and antigen or antibody and capture protein. The antibody will then be eluted off of the column or off of the bead and can be collected to complete the antibody purification process. If it is impossible for your experimental setup to immobilize the capture protein onto a solid support, then there is the option of using a fusion protein such as GST fusion protein (link) or a HIS-tag (6 histidin amino acids) fusion protein. These are protein chimeras that have a the GST or HIS tag and still retain the original shape and full function of the capture antibody. These fusion proteins can be incubated with the target antibody and then isolated from the solution via a solid support in a later step. The GST fusion protein will interact with glutathione on a column and is easily eluted with an excess of glutathione. The HIS tag is captured by a column loaded with immobilized metal ions such as cobalt, nickel, or copper. It is easily eluted by changing the buffer conditions.

The less stringent antibody purification methods can be used to collect monoclonal antibodies from a hybridoma cell line because the source is extremely controlled. However, polyclonal antibodies require the most stringent antigen-specific purification methods because there are so many proteins and antibodies in serum. Immunoassays are an important part of the biosciences, and they wouldn’t be possible without antibody purification strategies that produce consistent and trustworthy batches of antibodies.

 

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