Polyclonal Antibodies
Introduction
Polyclonal antibodies (pAbs) are complex mixtures of several antibodies produced by different clones of B cells in an animal. These antibodies recognize and bind to multiple distinct epitopes on an antigen, allowing them to form networks with the antigens. Each individual antibody recognizes a unique epitope present on the antigen. Polyclonal antibodies operate through a process known as antigen-antibody recognition. When an antigen enters the body, B cells identify specific regions known as epitopes on the surface of the antigen and bind to them. This activation of B cells leads to their differentiation into plasma cells, which then secrete antibodies, each with a unique binding specificity, into the bloodstream.
The diverse antibodies produced during the immune response allow polyclonal antibodies to target multiple epitopes on a specific antigen. This broad specificity enhances their ability to recognize and bind to different regions of the antigen, resulting in a higher success rate for identifying, neutralizing, and eliminating the foreign material.
Production of Polyclonal Antibodies
Preparation of Antigen
The quality and quantity of the antigen used directly impact the immune response. Even small amounts of impurities can lead to antibody reactions against contaminants rather than the desired antigen. An insufficient or excessive amount of antigen may cause sensitization, suppression, or other unintended immunomodulatory effects. Therefore, the purification of the antigen is a critical process for achieving enhanced antibody specificity.
The antigen must be prepared under sterile conditions to ensure it is free from endotoxins. The amount of antigen depends on various factors, including the specific properties of the antigen, the chosen animal species, the route of injection, the frequency of injections, and the purity level of the antigen.
Selection of Animal Species
Factors influencing the selection of animal species include the required amount of pAb, the phylogenetic relationship between the animal and the antigen, the age of the animal, ease of blood sampling, and the intended application of the pAb. Common laboratory animals used include rabbits, mice, guinea pigs, hamsters, goats, chickens, and sheep. Rabbits are preferred due to their size and relatively long lifespan. However, for producing larger quantities of pAbs, farm animals such as goats, sheep, and horses are used.
Immunization Protocol
The immunization protocol varies for different animal species. Adjuvants are compounds used as stimulants when the induced immune response is otherwise insufficient. The most commonly used adjuvant for producing pAbs is Freund’s complete adjuvant (FCA), which induces a high antibody titer against most types of antigens. However, care must be taken not to over-administer FCA, as excessive use can cause severe tissue damage.
The minimum volume of antigen capable of inducing an effective immune response is injected into the animal. The injection route depends on the nature of the antigen and the animal used. The antigen can be administered as a single large volume or as several smaller volumes at different injection sites.
If the antibody titer concentration reaches a plateau or begins to decline, a booster injection is administered. Such injections do not always require an adjuvant, and very small amounts of antigen may suffice to enhance antibody concentration. A maximum of three booster injections is recommended.
Post-Immunization Monitoring
Animals are monitored daily to assess any side effects of immunization, and blood samples are taken at regular intervals. The serum from the animals is analyzed to monitor antibody responses and to extract antibodies if sufficient quantities are produced.
Applications of Polyclonal Antibodies
Diagnostic Tests**: pAbs have a wide range of applications including diagnostic tests and quantitative and qualitative biological analyses. For example, pAbs are used in immunofluorescence and immunohistochemistry methods to identify tumor markers and other proteins of interest.
Therapeutic and Modulatory Purposes: pAbs are also used for mediating or modulating purposes such as immunotherapy, active signaling, or for neutralizing activities. An example is the use of pAbs in Digoxin Immune Fab for treating digoxin toxicity.
Prevention of Hemolytic Disease: pAbs like Rho(D) immunoglobulin are administered to Rh-negative mothers to prevent hemolytic disease in newborns. Rho(D) is produced from pooled human plasma collected from Rh-negative donors who have antibodies against the D antigen present on red blood cells.
Histopathological Analysis: pAbs are used in histopathological analyses employing immunoperoxidase staining. Beyond these applications, pAbs are utilized in immunoaffinity purification for the isolation or enrichment of antigens.
Recombinant pAbs: Due to their ability to target multiple tumor cell types compared to monoclonal antibodies, recombinant pAbs are used in cancer therapy. Monoclonal antibodies are widely used in cancer treatment, but disease recurrence due to the emergence of antibody-resistant tumor cells is common. By using pAbs, diverse recombinant antibodies can be generated that interact with various cancer types.
Future Directions: The path forward suggests using recombinant pAbs to minimize unnecessary cross-reactivity, as seen with traditional pAbs. The use of pAbs in various assays not only creates high throughput but may also be used to produce specific antibodies for human gene products that are renewable.
Conclusion
Polyclonal antibodies are generated using multiple different immune cells. They bind to the same antigen but recognize different epitopes, while monoclonal antibodies are produced from identical immune cells, all of which are clones of a single parent cell. For applications such as therapeutic drug production that require large volumes of identical antibodies specific to a single epitope, monoclonal antibodies are a better solution. However, for general research applications, the advantages of polyclonal antibodies often outweigh the specific benefits provided by monoclonal antibodies. The purification of serum binding affinity against small antigenic targets further enhances the advantages of polyclonal antibodies.
Antibody Purification
Antibodies are a critical component of the immune system. When the body is exposed to an antigen, it produces a specific antibody for that antigen. Techniques commonly used in biotechnology leverage this natural immune process. Antibodies are utilized in a variety of research applications as well as in immunoassays for disease detection. The antigen/antibody binding properties are used for immunogenicity detection and ELISA assays. We employ conjugated antibodies with fluorophores or enzyme labels to tag molecular targets on cells and entire tissues. Antibody purification is used for disease detection. These antibodies are typically obtained through antibody purification from humans, mice, rabbits, and chickens, depending on the application.
Polyclonal vs. Monoclonal Antibodies
The antibodies we use are derived from serum (polyclonal antibodies) or from hybridoma cell lines (monoclonal antibodies). Polyclonal antibodies are collected from different B cells, meaning there is greater diversity in recognizing antigen epitopes. Monoclonal antibodies are identical for a specific antigen and recognize the same epitope. The type of antibody desired depends on the objectives of the assay.
Three Main Methods for Antibody Purification
For antibody purification, it is essential to match the purification method with the type of antibody and the intended goal. There are three main methods for antibody purification. The most common method, which has less specificity in antibody purification, involves the simple separation of antibodies from other proteins based on size and structure. This method results in the purification of all antibodies regardless of type and antigen binding. The next step involves the selective isolation of a specific class of antibodies (e.g., IgG, IgM, IgA, IgL, IgE) using proteins such as Protein A or G that are dependent on specific classes of antibodies. The most specific method of antibody purification is antigen-specific adsorption, which often requires the antigen to be attached to a solid support. Magnetic beads are particularly useful for purifying antigen-specific antibodies and specific classes. Traditionally, columns or resin beads are used as solid supports, where antibodies are adsorbed to a specific antigen or protein and then collected. The binding of antibodies is most effective at physiological pH. If the pH is excessively raised or lowered, the binding between the antibody and antigen or the antibody and protein will be disrupted. The antibody is then washed from the column and can be collected to complete the antibody purification process. If the adsorbed protein cannot be fixed on a solid support, an option is to use fusion proteins or HIS6 tags (histidine amino acids).
Less stringent purification methods can be used to collect monoclonal antibodies from hybridoma cell lines, as this is a very controlled source. In contrast, polyclonal antibodies require the most stringent antigen-specific purification methods due to the presence of many proteins and antibodies in serum. Immunoassays are a crucial part of the life sciences, and they would not be feasible without antibody purification strategies that produce stable and reliable antibody batches.
General Methods for Antibody Purification
Physicochemical Fractionation Antibody Purification: In the physicochemical fractionation—differential precipitation method, immunoglobulins are separated based on size, charge, or other common chemical characteristics in typical samples, resulting in the isolation of a subset of proteins that include immunoglobulins.
Sub-methods of this approach include:
Size exclusion chromatography
Ammonium sulfate precipitation
Ion exchange chromatography
Immobilized metal chelate chromatography
Thiophilic adsorption
Melon Gel chromatography
Dialysis, desalting, and diafiltration can be used to exchange antibodies into specific buffers and remove unwanted low molecular weight (MW) components. Dialysis membranes, resins, and diafiltration devices with high molecular weight cut-off (MWCO) can be utilized to isolate immunoglobulins (> 140 kilodaltons) from smaller proteins and peptides. These techniques alone cannot purify antibodies from other proteins and macromolecules present in typical antibody samples, and purification is only possible using specialized columns and equipment. Typically, gel filtration and dialysis are followed by other purification steps, such as ammonium sulfate precipitation.
Ammonium sulfate precipitation is often used to enrich and concentrate antibodies from serum, ascitic fluid, or cell culture supernatant. Antibodies precipitate at lower concentrations of ammonium sulfate compared to other proteins and serum components. This purification method is suitable for certain antibody applications but is often performed as a preliminary step before column chromatography or other purification methods.
In ion exchange chromatography (IEC), positively or negatively charged resins are used to attach proteins based on their net charges in a specific buffer system (pH). IEC is a cost-effective, gentle, and reliable method for antibody purification.
Immobilized metal affinity chromatography (IMAC) uses divalent metal ions, such as nickel, to bind proteins or peptides that contain a histidine tag (often found in engineered recombinant proteins). Interestingly, mammalian IgGs are one of the few abundant proteins in serum (or hybridoma cell culture supernatant) that contain histidine amino acids, which can bind to immobilized nickel. Several other metal ions, such as cobalt, magnesium, zinc, calcium, and iron, are used as ligands in metal affinity chromatography.
Class-Specific Affinity Purification of Antibodies
Class-specific affinity purification employs solid-phase ligands that bind specific antibody classes (e.g., IgG) through biological ligands (proteins, lectins, etc.) with specific affinity for immunoglobulins. In this method, all antibodies of the target class are purified regardless of antigen specificity.
Protein A, G, and L Antibody-Binding Ligands
Proteins A, G, and L are three bacterial proteins with well-characterized antibody-binding properties. These proteins are produced recombinantly and are commonly used for affinity purification of key antibody types from various species. Most recombinant forms of these proteins that are commercially available have had their non-essential sequences removed, including the HSA binding domain from Protein G, making them smaller than their native counterparts. A genetically modified recombinant form of Protein A and Protein G known as Protein A/G is also widely available. All four recombinant Ig-binding proteins are commonly used by researchers in many diagnostic and immunological applications.
Sub-methods of this approach include:
Antibody Purification with Protein A, G, and L
IgM Purification
IgA Purification
Chicken IgY Purification
For antibody purification using Proteins A, G, A/G, or L, they are covalently attached to porous resins or magnetic beads. Since these proteins contain multiple binding domains for antibodies, nearly any immobilized molecule retains at least one functional, unobstructed binding domain regardless of orientation. Additionally, because the proteins bind to antibodies at sites other than the antigen-binding domain, they can be used in purification. Proteins A, G, A/G, and L exhibit different binding properties, making each suitable for various antibody targets. It is important to note that using Protein A, G, or L results in the purification of general immunoglobulins from a crude sample.
For IgM antibodies of class M that have the appropriate light chain type (VL-kappa), Protein L can be used for purification, although IgGs with the same light chain type are also purified. Industrial-scale and commercial purification for this class of antibody employs a combination of techniques.
Lectin is a glycoprotein weighing approximately 40 kilodaltons composed of four identical subunits. It is useful for purifying human serum or secretory IgA. The affinity ligand allows for the purification or removal of IgA from the more abundant IgG and IgM in human serum or colostrum.
Chickens produce a unique immunoglobulin molecule known as IgY. Chickens produce antibodies 15 to 20 times more than rabbits. An egg yolk from a vaccinated hen contains approximately 300 milligrams of IgY. Whole eggs or separated egg yolks can be collected and stored frozen for later antibody extraction.
Antigen-Specific Affinity Purification of Antibodies
In this method, all antibodies that bind to an antigen are purified regardless of the class or isotype of the antibody. Although Proteins A, G, A/G, and L are very important ligands for purifying total IgG from a sample, in many cases, antigen-specific antibody purification is required. In this method, specific antigens (used for immunization) are immobilized to purify those antibodies that specifically bind to that antigen.
Antigen Immobilization and Presentation
Peptide Antigens and Affinity Ligands
Protein Antigens and Affinity Ligands
Binding and Elution Conditions
The methods and degree of purity depend on the applications intended for the antibodies.