When a host animal is immunized with a prepared antigen during polyclonal antibody production, its immune system will respond by generating specific antibodies against that antigen to neutralize it and eliminate it from the body. Commercially produced polyclonal antibodies are available in unpurified serums that are created by sampling the blood of an immunized host animal and then removing clotting proteins and red blood cells.
This serum contains not only the antibody of interest but also several classes of immunoglobulins that react non-specifically to multiple target antigens and proteins as well as other serum proteins. The ability of these antibodies to recognize and bind to several different epitopes makes them vulnerable to cross-reactivity and background noise that can sometimes cause inaccurate or irreproducible data.
Purifying the polyclonal antibody serum before using it in an experiment is necessary to increase specificity (the ability of the antibody to differentiate between epitopes) and sensitivity (its ability to detect the presence of an antigen in a sample or generate “true positive” results).
This process eliminates unwanted proteins that could cause nonspecific cross-reactivity and enriches the desired antibodies that react to targeted antigens. Serum may be purified through protein A/G purification or antigen-specific affinity purification to improve the antibody performance in the intended application.
Protein A/G Purification
Antibodies have predictable structures with invariant domains. Because of this, researchers have identified specific protein ligands that bind to antibodies regardless of the antibody’s specificity to the target antigen.
Protein A and Protein G are two immunoglobulin-binding bacterial proteins that are produced recombinantly and are commonly used for purifying antibody types from a wide array of species. Protein A is a surface protein found on the cell walls of many strains of Staphylococcus aureus bacteria, and Protein G is a cell wall protein expressed in Group C and Group G strains of Streptococcus bacteria.
Protein A and Protein G are structurally similar, but they feature slightly different affinities for immunoglobulin G (IgG) subclasses across various animal species. Their affinities overlap, but Protein G tends to show a stronger affinity for rabbit, pig, cat, and dog IgG, while Protein G shows a stronger affinity for mouse and human IgG.
To prevent the risk of non-specific binding in assays, researchers have genetically engineered recombinant forms of Proteins A and G that have increased binding domains compared to their native forms, as well as a recombinant Protein A/G. Protein A/G contains four binding domains for Protein A and two binding domains for Protein A, allowing it to show an affinity for broader subclasses of IgG.
Protein A/G purification is ideal for purifying polyclonal antibodies generated against recombinant proteins or antigens. With purification involving a Protein A agarose resin column and rabbit serum, this method consists of the following steps:
- Covalently immobilizing the protein onto a porous resin column
- Adding a clarified, physiologic-buffered sample of rabbit serum to the column so the IgG can bind with the immobilized protein
- Washing away unbound serum components with a phosphate-buffered saline
- Adding an acidic elution buffer to dissociate the antibody from the immobilized protein
- Collecting small fractions of the purified IgG solution that pass through the column
- Using a protein assay or other tool for identifying and combining these fractions
- Neutralizing the buffer
Antigen-Specific Affinity Purification
While protein A/G purification does purify the total IgG from the raw serum and eliminate most of the unwanted serum proteins, the final preparation of the purified serum contains total non-specific IgG antibodies as well as antibodies specific to the target antigen. This means it still possesses the potential for cross-reactivity and additional purification of the antigen-specific antibodies is frequently required.
Researchers use antigen-specific affinity purification to isolate and enrich the antibodies specific to the antigen to remove the unwanted antibodies from the preparation. With this method, researchers can obtain the purest polyclonal antibodies with high specificity and sensitivity and low cross-reactivity. Immunohistochemistry and immunofluorescence experiments benefit from this type of purification because the antibodies can be used at lower dilutions without increasing background noise.
Antigen-specific affinity purification is comprised of the following steps:
- Isolating the IgG fraction of the antibody population through ammonium sulfate precipitation to remove sticky proteins and provide a cleaner preparation
- Preparing an affinity resin by cross-linking the antigen (typically a peptide) to a carrier protein
- Immobilizing target antigen-specific antibodies to the resin with the same chemistry used in the cross-linking so all epitopes are available for binding
- Passing the serum through an antigen affinity column matrix (the antigen-specific antibody will remain bound to the column)
- Purifying the antigen-specific antibodies with pH gradient elution to eliminate most of the remaining non-specific immunoglobulin fraction
- Collecting the antibodies in a neutralizing buffer
- Concentrating and measuring the purified antibodies
- Performing ELISA to confirm specificity for the target and determine antibody titer