Antibody purification methods

Introduction to antibody formats and purification

Antibody purification is a vital step for ensuring consistent assay results and it also plays a major role in excluding non-specific binding and allowing the end product to be used for different detection methods. Furthermore, the purification process allows the antibodies to be tagged with an enzyme or a fluorophore, expanding their uses in a variety of assays.

The role of the purification step is the selective enrichment and specific antibody isolation from serum for polyclonal antibodies or from ascites fluid and hybridoma cell line when working with monoclonal antibodies. Over the years, many different methods have been established, some boasting high specificity and others being cruder. In general, they have been classified as physiochemical fractionation, class-specific affinity and antigen-specific affinity.

The biggest difference between monoclonal and polyclonal antibodies regarding purification is that the latter requires antigen-specific purification to inhibit co-purification of non-specific IgG's. When purifying monoclonal antibodies from ascites fluid or cell culture supernatant, using antigen-specific purification is not necessary, because the antibody of interest is the only one that can be found in the sample.

Physicochemical fractionation antibody purification

The key immunoglobulin classes, IgG and IgM have the same overall structure and amino acid composition, making these classes relatively different from other proteins found in serum, like albumin and transferrin.

These physiochemical differences are used to select and separate the target immunoglobulins from the unwanted proteins found in serum using size-exclusion or ion-exchange chromatography and ammonium sulfate precipitation among other methods.

Size exclusion chromatography

Desalting and dialysis are used to separate low-molecular-weight components from the target antibodies in a suitable buffer. This is possible due to immunoglobulins usually having a molecular weight of over 140kDa, allowing for easy separation using dialysis membranes and diafiltration devices with high-molecular-weight cut-offs, thus limiting the exchange of small proteins and peptides.

The downside of this method is that unless highly specialised equipment is used, full purification from proteins and macromolecules cannot be achieved by only using size exclusion chromatography and often this method is used following ammonium sulfate precipitation as an additional step.

Ammonium sulfate precipitation

This method of antibody purification is often used for both monoclonal and polyclonal samples. Ammonium sulfate precipitation relies on the different solubility of proteins and antibodies. With the concentration of the lyotropic salt progressively increasing in the sample, the antibodies are the first to become less soluble and eventually precipitate, with this lyotropic effect called "salting out".

Ammonium salt precipitation is can provide good purification for different antibody applications but to achieve better results it is more commonly used in conjunction with column chromatography to improve the sample performance.

Ion exchange chromatography

Ion exchange chromatography is one of many different chromatography methods that are used for antibody purification. This assay uses negatively or positively charged resins for protein binding in a buffer based on their charges.

IEC can be often found in monoclonal antibody production, where the conditions can be optimised so the target immunoglobulin is bound and released with very high specificity, making it a reliable, cost-effective and gentle method of antibody purification.

Class-specific affinity purification

Due to the mostly invariable domains and native function of antibodies, some pathogenic bacteria have evolved proteins with specific binding functions. The native anti-Ig proteins have been isolated and are used as affinity ligands in antibody purification.

One of the most well-researched and commonly used proteins for affinity purification are proteins A, G, and L, derived from the S. aureus, group C and G Streptococcus spp. and P. magnus bacteria. The antibody-binding properties of these three bacterial proteins are essential for using them in purification methods in crude samples.

To expand their uses, the recombinant forms of these proteins have had their unnecessary sequences removed to make them more compact and a recombinant combination of protein A and G has been successfully engineered and has a vital role in a variety of immunodetection and immunoaffinity assays.

This method of purification removes the majority of the serum proteins but it cannot clear the sample of non-specific immunoglobulin fractions, resulting in possible cross-reactivity.

Antigen-specific affinity purification

Total IgG purification can be easily obtained by using the previously described proteins A, G, L, and A/G but the downside of this method is that it cannot purify them based on their antigen specificity.

To perform this additional step, the antigen used in the immunisation needs to be immobilised so only the antibodies that bind it gets purified.