Antibodies are a crucial part of the body’s immune system. Upon getting infected with an antigen, the immune system generates an antibody response specific to that antigen. Antibodies are used in many biomedical research applications as well as in immunoassays for disease detection. Thus, the procedure of antibody purification is an essential research procedure in infectious disease and immunology research, where primary cells play the role of an excellent model. In this article, we discuss some conventional strategies for antibody purification:

Conventional Antibody Purification Strategies

Antibody purification strategy has been popularly confined to Protein A, Protein G, and Protein L beads in chromatography through the binding capacity depends on antibody class. Protein A and G beads are more commonly used than Protein L beads for human IgG but not efficient enough to isolate human IgD, IgA, IgM, and IgE.

Protein A beads

Protein A is a cell wall component of Staphylococcus aureus strains. It is a 42kDa single polypeptide chain covalently coupled to beaded agarose gel. This can specifically bind to the Fc region of immunoglobulin molecules. These beads have a special affinity for IgG of several mammalian species but the binding efficiency difference of Protein A to sub-classes of IgG allows separate IgG type binding. The antibodies that do not bind to Protein A can be recovered from the Flow-Through fractions.

Protein G beads

Protein G is 22kDa cell wall protein from group G streptococci. The Ig-binding sites of protein A and protein G are very different though tertiary structures are similar, as there are changes in amino acid composition. Protein G can bind Fc and Fab regions of Ig from various species.

Protein L beads

Protein L is a 35.8kDaIg-binding protein from the bacteria Peptostreptococcus Magnus. It binds the k light chain of the VL domains of different Ig (IgG, IgA, IgM, IgD, IgE, and IgY) with different binding affinities. The important criteria of this binding depend on the presence of a K light chain in immunoglobulin molecules. Examples of Protein L beads are Capto L (GE) which can capture IgG and IgM Ab fragments with high efficiency.

In most of these cases, classical protein beads or resins are effective for antibody capturing but for certain species, fragments, or proteins of interest, it becomes difficult to isolate with conventional strategies. Therefore, it is important to develop innovative customized strategies for optimizing antibody purification.

Antibody purification is a complicated and calculated science that poses many challenges to researchers who are working on protein engineering research. As cell culture goes for a scale-up, researchers have to come up with more strategies of antibody purification or design new ones. If you are working on primary cell culture for antibody production, make sure the initial steps ensure research efficacy before you develop purification strategies.

For more information on research strategies and primary cell culture expertise, contact info@kosheeka.com with your inquiries.

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