Antibody Purification Methods
This guide will give an overview of antibody purification methods. Antibodies that are directed toward a specific target protein is instrumental in the fields of bioresearch and diagnostic technologies. Animals immunized with prepared antigens will produce specific antibodies against the antigen. Once they are purified (and possibly after labeling them with an enzyme or fluorescent tag), these antibodies can be used directly to probe the specific antigen in Western blotting, ELISA and other applications.
Antiserum from an immunized animal can be used directly for certain applications, but more often some form of antibody purification is required to obtain an antibody probe that is effective for multiple types of detection methods. This article summarizes the main approaches and tools available for accomplishing antibody purification.
There are multiple ways to purify antibodies. Each method has its own advantages and disadvantages. All purification methods involve an antibody enrichment or specific isolation of antibodies from serum (polyclonal antibodies), ascites fluid/cell culture supernatant of a hybridoma cell line (monoclonal antibodies). Purification can be range from very crude to highly specific. The intended use for the antibody should be considered when selecting the best purification method. Three antibody classifications are:
- Fractionation Purification– is the differential precipitation, size-exclusion or solid-phase binding of immunoglobulins based on size, charge or other shared chemical characteristics of antibodies in typical samples. This isolates a subset of sample proteins that includes the immunoglobulins.
- Class-specific AffinityPurification– another antibody purification method is the solid-phase binding of particular antibody classes (e.g., IgG) by immobilized biological ligands (proteins, lectins, etc.) that have specific affinity to immunoglobulins. This purifies all antibodies of the target class without regard to antigen specificity.
- Antigen-specific Affinity Purification– affinity purification of only those antibodies in a sample that bind to a particular antigen molecule through their specific antigen-binding domains. This purifies all antibodies that bind the antigen without regard to antibody class or isotype.
Monoclonal antibody hybridoma cell lines produced as ascites fluid or cell culture supernatant can be fully purified without using an antigen-specific affinity method. The target antibody is the only immunoglobulin in the production sample. Polyclonal antibodies from antiserum have many circulation immunoglobulins and require antigen-specific affinity purifications to prevent co-purification of nonspecific immunoglobulins. IR Tip- Only ~2-5% of total IgG in antiserum is specific for the antigen used to immunize the animal.
Fractionation Antibody Purification
The main classes of serum immunoglobulins are IgG, IgM, IgA, IgE, IgD. The classes share the same general structure, including overall amino acid composition and solubility characteristics. These properties are sufficiently different from most other abundant proteins in serum, such as albumin and transferrin. This allows them to be selected and enriched on the basis of their differentiating physicochemical properties.
Ammonium Sulfate Precipitation
Ammonium sulfate precipitation is one of the most frequently used antibody purification method to enrich and concentrate antibodies from serum, ascites fluid or cell culture supernatant. Proteins will precipitate out of solution at varying salt concentrations. As the concentration of salt is increased proteins and other macromolecules become progressively less soluble until they precipitate. This is sometimes referred to as “salting out.” Antibodies are unique in that they precipitate at lower concentrations of ammonium sulfate than most other proteins and components of serum. This allows them to precipitate first and be captured. The results are sometimes called an Ig Fraction. IR TIP- Many protocols call for using solid granular Ammonium Sulfate. This is not recommended as it produce zones of high concentration while it dissolves in the sample. Start by make a supersaturated Ammonium Sulfate solution.
Typically immunoglobulins precipitate between 40% and 50% ammonium sulfate saturation. The usual method involves very slowly adding an equal volume of saturated ammonium sulfate solution to a neutralized antibody sample, followed by incubation for several hours at room temperature or 4°C. After centrifugation and removal of the supernatant, the antibody-pellet is dissolved in buffer, such as phosphate-buffered saline (PBS).
The selectivity, yield, purity and reproducibility of precipitation depends upon several factors, including time, temperature, pH and rate of salt addition. Ammonium sulfate precipitation provides sufficient purification for some antibody applications, but most often it is performed as a preliminary step before column chromatography or other purification method. Using partially-purified antibody samples can improve the performance and extend the life of affinity columns.
Ion Exchange Chromatography
Numerous chemically-based, solid-phase chromatography methods have been adapted and optimized to achieve antibody purification in particular situations.
Ion exchange chromatography (IEC) uses positively or negatively charged resins to bind proteins based on their net charges in a given buffer system (pH). Especially in commercial operations involving production of monoclonal antibodies, conditions for IEC can be determined that bind and release the target antibody with a high degree of specificity. Conversely, conditions can be found that bind nearly all other sample components except antibodies. Once so optimized, IEC is a cost-effective, gentle and reliable method for antibody purification.
Size Exclusion Chromatography (SEC)
Dialysis, desalting and diafiltration can be used to exchange antibodies into particular buffers and remove undesired low-molecular weight (MW) components. Dialysis membranes, size-exclusion resins, and diafiltration devices that feature high-molecular weight cut-offs (MWCO) can be used to separate immunoglobulins (>140kDa) from small proteins and peptides. However, except with specialized columns and equipment, these techniques alone cannot purify antibodies from other proteins and macromolecules that are present in typical antibody samples. More commonly, gel filtration and dialysis are used as a “polishing” step, such as ammonium sulfate precipitation .
Immobilized Metal Chelate Chromatography
Immobilized metal chelate chromatography (IMAC) uses chelate-immobilized divalent metal ions (usually nickel, Ni2+) to bind proteins or peptides that contain clusters of three or more consecutive histidine residues. The strategy is most often used to purify recombinant proteins that have been engineered to contain a terminal 6xHis fusion tag. Interestingly, mammalian IgGs are one of the few abundant proteins in serum (or monoclonal hybridoma cell culture supernatant) that possess histidine clusters capable of being bound by immobilized nickel. As with IEC, IMAC conditions for binding and elution can be optimized for particular samples to provide gentle and reliable antibody purification. For example, the Pierce Conjugate Purification Kit uses this technique to separate AP- or HRP-labeled (enzyme-conjugated) antibody from excess, non-conjugated enzyme following a labeling procedure.
Class-specific Affinity Purification of Antibodies
Because antibodies have a conserved overall structure certain pathogenic bacteria have evolved proteins having specific antibody-binding functions. Several of these immunoglobulin-binding proteins have been identified and isolated from certain species of bacteria. In the same way that the native functions of antibodies are useful as target-specific probes for protein research, so also these native anti-Ig proteins are useful as affinity ligands for antibody purification.
Protein A, G, A/G and L Antibody-binding Ligands
Protein A, Protein G and Protein L are three bacterial proteins whose antibody-binding properties have been well characterized. These proteins have been produced recombinantly and used routinely for affinity purification of key antibody types from a variety of species. Most commercially-available, recombinant forms of these proteins have unnecessary sequences removed. A genetically-engineered recombinant form of Protein A and Protein G, called Protein A/G, is also widely available. These are commonly used in antibody purification methods. All four recombinant Ig-binding proteins are used routinely by researchers in numerous immunodetection and immunoaffinity applications.
Sources and features of native Ig-binding proteins.
|Protein A (SpA)||Protein G (SpG)||Protein L (SpL)|
(Group C and G)
|Component of human body flora; cause of “Staph” infections||Orig. isolated from pharyngitis patients (tonsils or blood)||Commensal and/or pathogenic anaerobic Gram-positive bacteria|
|40 to 60kDa
(variable numbers of repeated domains)
|40 to 65kDa
(variable numbers of repeated domains)
|Binding Domains||5 for IgG (most common form)||1 to 2 for IgG
0 to 2 for HSA
|5 for Ig|
|Ig-binding Target||heavy chain constant region (Fc) of IgG (CH2-CH3 region)||heavy chain const. region (Fc) of IgG (CH2-CH3 region)||kappa light chains of Igs (VL-kappa)|
Antibody Purification with Protein A, G and L
Antibody purification with Protein A, Protein G, Protein A/G or Protein L, the protein is covalently immobilized onto porous resins (such as beaded agarose) or magnetic beads. Because these proteins contain several antibody-binding domains, nearly every individual immobilized molecule, no matter its orientation maintains at least one functional and unhindered binding domain. Furthermore, because the proteins bind to antibodies at sites other than the antigen-binding domain, the immobilized forms of these proteins can be used in purification schemes, such as immunoprecipitation, in which antibody binding protein is used to purify an antigen from a sample by binding an antibody while it is bound to its antigen.
Protein A, G, A/G and L have different binding properties, which make each one suitable for different types of antibody targets (e.g., antibody subclass or animal species). It is important to realize that use of Protein A, G or L results in purification of general immunoglobulin from a crude sample. Depending on the sample source, antigen-specific antibody may account for only a small portion of the total immunoglobulin in the sample. For example, generally only 2-5% of total IgG in mouse serum is specific for the antigen used to immunize the animal.
Using a column of Protein A agarose resin and rabbit serum as the example, the general procedure for antibody purification with these ligands is as follows:
- Bind:Add a clarified, physiologic-buffered (pH 7 to 8) sample of rabbit serum to the column and allow it to slowly pass through or mix with the Protein A resin to allow the IgG to bind to the immobilized ligand.
- Wash:Add phosphate-buffered saline (PBS) and allow it to pass through the column to wash away nonbound serum components. Use a volume of wash buffer equivalent to 5 to 10 times the resin volume.
- Elute:Add acidic elution buffer (e.g., 0.1M glycine-HCl, pH 2.8), and collect small fractions of solution that pass from the column. The low-pH condition dissociates the antibody from the immobilized Protein A, and the IgG is recovered in its purified state in one or several of the collected fractions.
- Neutralize or exchange buffer:Use a protein assay or other means to identify and combine elution fractions that contain the purified antibody. Then add 1/10th volume of 1M Tris-HCl (pH 8.5) to neutralize the buffer. Alternatively, use a desalting column or dialysis to exchange the purified antibody into a more suitable buffer for long-term storage.
Protein A and Protein G bind IgM very poorly, in part because binding sites on the Fc regions of IgM are sterically hindered by its pentameric structure. For large scale operations, IgM antibodies are usually purified by a combination of techniques, including ammonium sulfate precipitation followed by gel filtration, ion exchange chromatography or zone electrophoresis. With serum samples (polyclonal), a simple enrichment strategy is ammonium sulfate precipitation followed by removal of IgG with Protein A or G.
Jacalin is a D-galactose binding lectin extracted from jackfruit seeds. The lectin is a 40kDa glycoprotein of composed of four identical subunits. Jacalin can be immobilized on agarose beads and has been useful for the purification of human serum or secretory IgA1. The affinity ligand makes it possible to purify or remove IgA from the more abundant IgG and IgM in human serum or colostrum.
Chicken IgY Purification
Chickens produce a unique immunoglobulin molecule called IgY. There are several advantages to production and use of IgY over mammalian immunoglobulins. With regard to production, raising and immunizing chickens is relatively simple, chickens are more likely to produce an immune response to conserved mammalian protein antigens, and chickens produce 15- to 20-times more antibody than rabbits.
Most importantly, IgY is naturally packaged at high concentrations in egg yolks, making repeated collection of antibody from immunized hens noninvasive. A single egg yolk from an immunized chicken contains approximately 300mg of IgY. Whole eggs or separated egg yolks can be collected and stored frozen for later extraction of antibody.
Protein A, Protein G and other Fc-binding proteins do not bind IgY. Most IgY purifications methods uses a variation of ammonium sulfate precipitation after first delipidating the egg yolk sample with various delipidation agents.
Antigen-specific Affinity Purification of Antibodies
Although Proteins A, G, A/G and L are excellent ligands for purification of total IgG from a sample, purification of antigen-specific antibodies is often required. This can be accomplished by immobilizing the particular antigen used for immunization so that only those antibodies that bind specifically to the antigen are purified in the procedure. Activated affinity supports that can be used to immobilize peptides or other antigens.
Antigen Immobilization and Presentation
Successful affinity purification of antibody depends on effective presentation of the relevant epitopes on the antigen to binding sites of the antibody. If the antigen is small and immobilized directly to a solid support surface by multiple chemical bonds, important epitopes may be blocked or sterically hindered, prohibiting effective antibody binding. Therefore, it is best to immobilize peptide antigens using a unique functional group (e.g., sulfhydryl on a single terminal cysteine in a peptide) and to use an activated support whose reactive groups occur on spacer arms that are several atoms long. For large (e.g., protein) antigens, especially those with multiple sites of immobilization, the spacer arm length becomes less important since the antigen itself serves as an effective spacer between the support matrix and the epitope. Generally, if the antigen was crosslinked to a carrier protein to facilitate antibody production, best results are obtained when the antigen is immobilized for affinity purification using the same chemistry (e.g., reaction to primary amines, sulfhydryls, carboxylic acids or aldehydes). In this way, all epitopes will be available for antibody binding, allowing greater efficiency in purification and recovery of the specific immunoglobulin.
Peptide Antigens and Affinity Ligands
Most antibodies are produced using peptide antigens that were synthesized and conjugated to an immunogenic carrier protein, such as KLH or BSA. Such antigens can be customized to contain a unique functional group (handle) for both conjugation and immobilization. A terminal cysteine is often added for this purpose; it provides a sulfhydryl group for efficient conjugation to maleimide-activated carrier protein and immobilization onto iodoacetyl-activated agarose resin (SulfoLink Coupling Resin).
Another common strategy is to use amine-functionalized resin and EDC crosslinker to immobilize peptides via their carboxyl (C-terminal) amino acids. Because peptides have both amine and carboxyl termini (as well as possibly internal lysines, aspartate or glutamate residues), the carboxyl-to-amine crosslinking caused by EDC boths polymerize and immobilizes the peptide so that it is presented to the antibody in a variety of orientations for affinity binding.
Protein Antigens and Affinity Ligands
Except in special situations, protein antigens are usually most easily immobilized for affinity purification by targeting primary amines, which typically occur in several locations at the outer surface of protein structures (i.e., wherever there are lysine groups or an N-terminus of a subunit). Several high-capacity, amine-reactive affinity supports are available for this sort of immobilization.
Alternatively, if the protein antigen is a purified glycoprotein and the carbohydrate groups are not the epitopes of interest, then the antigen can be covalently immobilized through the sugar groups after they have been oxidized with sodium periodate.
Binding and Elution Conditions
Little variation exists among typical binding and elution conditions for antigen-specific affinity purification of antibodies because they are based on native affinity interactions of antibodies with their respective antigens. That is, because antibodies are designed to recognize and bind antigens tightly under physiologic conditions, most affinity purification procedures use binding conditions that mimic physiologic pH and ionic strength. The most common binding buffers are phosphate-buffered saline (PBS) and Tris-buffered saline (TBS) at pH 7.2. Once the antibody has been bound to an immobilized antigen, additional binding buffer is used to wash unbound material from the support. To minimize nonspecific binding, many researchers use wash buffer containing additional salt or detergent to disrupt any weak interactions.
Specific, purified antibodies can be eluted from an affinity resin by sufficiently altering the pH or ionic strength of the buffer to disrupt the antigen-binding interaction. Most antibodies are moderately resilient proteins that tolerate a range of pH from 2.5 to 11.5 without permanent inactivation, and low-pH is by far the most common elution strategy. In some cases an antibody-antigen interaction is not efficiently disrupted by pH changes or is damaged by the pH, requiring that an alternate strategy be used.
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