Purification of antibodies involves separating them from serum (polyclonal antibodies), ascites fluid, or the culture supernatant of hybridoma cells (monoclonal antibodies). The purification methods can be crude or highly specific.
The purification level necessary to make usable antibodies depends on the intended applications. You can purify antibodies from immobilized antigens or by class-specific purification. A good example is when IgG is not subject to specific consideration.
Over time, antibodies have changed in structure. The following are some antibody purification methods by use of Elisa Kits and others.
Protein A, G, and L
These proteins bind to specific immunoglobulins. They are bacterial proteins with specific domains. Proteins A and G bind to the Fc region, while protein L binds to the light chain of the Fab region.
The IgD molecule binds strongly to protein A, whereas protein G weakly binds to IgA and IgM. Protein G binds only to IgG, while protein L binds to all antibody classes. Using these bacterial proteins, it is, therefore, possible to separate specific immunoglobulin classes.
You can use a chromatography column containing protein A as an example, where protein A binds to serum and separates it from IgG.
Purification of IgM
Proteins poorly bind IgM A and G, in part because their pentameric structure prevents their binding sites from interacting with FC regions. Protein L can purify IgMs (class M antibodies) containing the light chains (VL-kappa), but this method lso applies to IgGs containing the same light chains.
It purifies IgM antibodies using multiple methods, including ammonium sulfate precipitation followed by gel filtration, ion exchange chromatography, or zone electrophoresis for commercial operations. A simple enrichment strategy for serum samples (polyclonal) is ammonium sulfate precipitation followed by removal of IgG with Protein A or G.
Purification of mouse IgM from ascites is most effective with the IgM Purification Kit using immobilized MBP. Using an affinity column, you can get purified IgM in just one pass. As determined by HPLC, human IgM will bind to support at least 88% purified, albeit with a slightly decreased capacity.
Purification of IgA
Jacalin, a D-galactose lectin isolated from jackfruit seeds, revealed this method. It binds to IgA and comprises four identical domains. Immobilizing jacalin on agarose gels and then separating IgA from other immunoglobulins is achievable using a method known as fractionation.
Purification of IgY
Chickens produce a unique immunoglobulin molecule called IgY. In contrast with mammalian immunoglobulins, IgY has several advantages. In terms of production, raising and immunizing chickens is relatively simple, and chickens are 15- to 20-times more likely to produce antibodies to conserved mammalian protein antigens than rabbits.
The naturally occurring IgY found in egg yolks makes it possible to collect antibodies from immunized chickens without invasive methods. A chicken immunized with IgY has about 300mg of IgY in its yolk. You can freeze egg yolks or whole eggs to work with antibodies.
To purify antibodies, it is most practical and cost-effective to first separate large contaminants by physiochemical processes, including aqueous two-phase separations, followed by a finer, more specific separation by chromatography.
Producing a generic antibody purification platform would be ideal. This approach may prove infeasible or quite difficult given the inherent antibody diversity and the generation of increasingly complex derivatives of antibodies.