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Polyclonal antibodies (pAbs) are antibodies that are secreted by different B cell lineages within the body (whereas monoclonal antibodies come from a single cell lineage). They are a collection of immunoglobulin molecules that react against a specific antigen, each identifying a different epitope.
The general procedure to produce polyclonal antibodies is as follows:
An antigen/adjuvant conjugate is injected into an animal of choice to initiate an amplified immune response. After a series of injections over a specific length of time, the animal is expected to have created antibodies against the conjugate. Blood is then extracted from the animal and then purified to obtain the antibody of interest.
Inoculation is performed on a suitable mammal, such as a mouse, rabbit or goat. Larger mammals are often preferred as the amount of serum that can be collected is greater. An antigen is injected into the mammal. This induces the B-lymphocytes to produce IgG immunoglobulins specific for the antigen. This polyclonal IgG is purified from the mammal's serum.
By contrast, monoclonal antibodies are derived from a single cell line.
Many methodologies exist for polyclonal antibody production in laboratory animals. Institutional guidelines governing animal use and procedures relating to these methodologies are generally oriented around humane considerations and appropriate conduct for adjuvant (agents which modify the effect of other agents while having few if any direct effects when given by themselves) use. This includes adjuvant selection, routes and sites of administration, injection volumes per site and number of sites per animal. Institutional policies generally include allowable volumes of blood per collection and safety precautions including appropriate restraint and sedation or anesthesia of animals for injury prevention to animals or personnel.
The primary goal of antibody production in laboratory animals is to obtain high titer, high affinity antisera for use in experimentation or diagnostic tests. Adjuvants are used to improve or enhance an immune response to antigens. Most adjuvants provide for an injection site, antigen depot which allows for a slow release of antigen into draining lymph nodes.
Many adjuvants also contain or act directly as:
Such antigens by themselves are generally poor immunogens. Most complex protein antigens induce multiple B-cell clones during the immune response, thus, the response is polyclonal. Immune responses to non-protein antigens are generally poorly or enhanced by adjuvants and there is no system memory.
Antibodies are currently also being produced from isolation of human B-lymphocytes to produce specific recombinant monoclonal antibody mixtures. The biotechnology company, Symphogen, develops this type of antibodies for therapeutic applications. They are the first research company to reach phase two trials with the monoclonal antibody mixtures that mimic the diversity of the polyclonal antibody drugs. This production prevents viral and prion transmission and this is the simple process.
Animals frequently used for polyclonal antibody production include chickens, goats, guinea pigs, hamsters, horses, mice, rats, and sheep. However, the rabbit is the most commonly used laboratory animal for this purpose. Animal selection should be based upon:
Goats or horses are generally used when large quantities of antisera are required. Many investigators favor chickens because of their phylogenetic distance from mammals. Chickens transfer high quantities of IgY (IgG) into the egg yolk and harvesting antibodies from eggs eliminates the need for the invasive bleeding procedure. One week's eggs can contain 10 times more antibodies than the volume of rabbit blood obtained from one weekly bleeding. However, there are some disadvantages when using certain chicken derived antibodies in immunoassays. Chicken IgY does not fix mammalian complement component C1 and it does not perform as a precipitating antibody using standard solutions.
Although mice are used most frequently for monoclonal antibody production, their small size usually prevents their use for sufficient quantities of polyclonal, serum antibodies. However, polyclonal antibodies in mice can be collected from ascites fluid using any one of a number of ascites producing methodologies.
When using rabbits, young adult animals (2.5–3.0 kg or 5.5–6.5 lbs) should be used for primary immunization because of the vigorous antibody response. Immune function peaks at puberty and primary responses to new antigens decline with age. Female rabbits are generally preferred because they are more docile and are reported to mount a more vigorous immune response than males. At least two animals per antigen should be used when using outbred animals. This principle reduces potential total failure resulting from non-responsiveness to antigens of individual animals.
The size, extent of aggregation and relative nativity of protein antigens can all dramatically affect the quality and quantity of antibody produced. Small polypeptides (<10 ku) and non-protein antigens generally need to be conjugated or crosslinked to larger, immunogenic, carrier proteins to increase immunogenicity and provide T cell epitopes. Generally, the larger the immunogenic protein the better. Larger proteins, even in smaller amounts, usually result in better engagement of antigen presenting antigen processing cells for a satisfactory immune response. Injection of soluble, non-aggregated proteins has a higher probability of inducing tolerance rather than a satisfactory antibody response.
Keyhole limpet hemocyanin (KLH) and bovine serum albumin are two widely used carrier proteins. Poly-L-lysine has also been used successfully as a backbone for peptides. Although the use of Poly-L-lysine reduces or eliminates production of antibodies to foreign proteins, it may result in failure of peptide-induced antibody production. Recently, liposomes have also been successfully used for delivery of small peptides and this technique is an alternative to delivery with oily emulsion adjuvants.
Selection of antigen quantity for immunization varies with the properties of the antigen and the adjuvant selected. In general, microgram to milligram quantities of protein in adjuvant are necessary to elicit high titer antibodies. Antigen dosage is generally species, rather than body weight, associated. The so-called “window” of immunogenicity in each species is broad but too much or too little antigen can induce tolerance, suppression or immune deviation towards cellular immunity rather than a satisfactory humoral response. Optimal and usual protein antigen levels for immunizing specific species have been reported in the following ranges:
Optimal “priming” doses are reported to be at the low end of each range.
The affinity of serum antibodies increases with time (months) after injection of antigen-adjuvant mixtures and as antigen in the system decreases. Widely used antigen dosages for “booster” or secondary immunizations are usually one half to equal the priming dosages. Antigens should be free of preparative byproducts and chemicals such as polyacrylamide gel, SDS, urea, endotoxin, particulate matter and extremes of pH.
When a peptide is being used to generate the antibody, it is extremely important to design the antigens properly. There are several resources that can aid in the design as well as companies that offer this service. Expasy has aggregated a set of public tools under its ProtScale page that require some degree of user knowledge to navigate. For a more simple peptide scoring tool there is a Antigen Profiler tool available that will enable you to score individual peptide sequences based upon a relation epitope mapping database of previous immunogens used to generate antibodies. Finally, as a general rule peptides should follow some basic criteria.
When examining peptides for synthesis and immunization, it is recommended that certain residues and sequences be avoided due to potential synthesis problems. This includes some of the more common characteristics:
Investigators should also consider the status of nativity of protein antigens when used as immunogens and reaction with antibodies produced. Antibodies to native proteins react best with native proteins and antibodies to denatured proteins react best with denatured proteins. If elicited antibodies are to be used on membrane blots (proteins subjected to denaturing conditions) then antibodies should be made against denatured proteins. On the other hand, if antibodies are to be used to react with a native protein or block a protein active site, then antibodies should be made against the native protein. Adjuvants can often alter the nativity of the protein. Generally, absorbed protein antigens in a preformed oil-in-water emulsion adjuvant, retain greater native protein structure than those in water-in-oil emulsions.
Antigens should always be prepared using techniques that ensure that they are free of microbial contamination. Most protein antigen preparations can be sterilized by passage through a 0.22 μm filter. Septic abscesses often occur at inoculation sites of animals when contaminated preparations are used. This can result in failure of immunization against the targeted antigen.
There are many commercially available immunologic adjuvants. Selection of specific adjuvants or types varies depending upon whether they are to be used for research and antibody production or in vaccine development. Adjuvants for vaccine use only need to produce protective antibodies and good systemic memory while those for antiserum production need to rapidly induce high titer, high avidity antibodies. No single adjuvant is ideal for all purposes and all have advantages and disadvantages. Adjuvant use generally is accompanied by undesirable side effects of varying severity and duration. Research on new adjuvants focuses on substances which have minimal toxicity while retaining maximum immunostimulation. Investigators should always be aware of potential pain and distress associated with adjuvant use in laboratory animals.
The most frequently used adjuvants for antibody production are Freund's, Alum, the Ribi Adjuvant System and Titermax.
Digoxin Immune Fab is the antigen binding fragment of polyclonal antibodies raised to Digitalis derivative as a hapten bound to a protein and is used for the reversal of life-threatening digoxin or digitoxin toxicity. [1] [2] [3]
Rho(D) immune globulin is made from pooled human plasma provided by Rh-negative donors with antibodies to the D antigen. It is used to provide passive immune binding of antigen, preventing a maternal active immune response which could potentially result in hemolytic disease of the newborn. [4] [5]
Rozrolimupab is the anti-RhD recombinant human polyclonal antibody composed of 25 unique IgG1 antibodies and is used for the treatment of immune thrombocytopenia purpura and prevention of isoimmunization in Rh-negative pregnant women.
REGN-COV2 (Regeneron Pharmaceuticals) – potential treatment for people with COVID-19 and to prevent SARS-CoV-2 coronavirus infection.
The use of polyclonal antibodies (PAbs) over monoclonal antibodies has its advantages. The technical skills needed to produce polyclonal antibodies is not as demanding. They're inexpensive to make and can be generated fairly quickly, taking up to several months to produce. PAbs are heterogeneous, which allows them to bind to a wide range of antigen epitopes. Because PAbs are produced from a large number of B cell clones, they're more likely to successfully bind to a specific antigen. PAbs remain stable in different environments, such as a change in pH or salt concentration, which allows them to be more applicable in certain procedures. Additionally, depending on the amount needed, PAbs can be made in large quantities in relation to the size of the animal used. [6] [7] [8]
In immunology, an antigen (Ag) is a molecule, moiety, foreign particulate matter, or an allergen, such as pollen, that can bind to a specific antibody or T-cell receptor. The presence of antigens in the body may trigger an immune response.
An antibody (Ab) is the secreted form of a B cell receptor; the term immunoglobulin (Ig) can refer to either the membrane-bound form or the secreted form of the B cell receptor, but they are, broadly speaking, the same protein, and so the terms are often treated as synonymous. Antibodies are large, Y-shaped proteins belonging to the immunoglobulin superfamily which are used by the immune system to identify and neutralize antigens such as bacteria and viruses, including those that cause disease. Antibodies can recognize virtually any size antigen with diverse chemical compositions from molecules. Each antibody recognizes one or more specific antigens. Antigen literally means "antibody generator", as it is the presence of an antigen that drives the formation of an antigen-specific antibody. Each tip of the "Y" of an antibody contains a paratope that specifically binds to one particular epitope on an antigen, allowing the two molecules to bind together with precision. Using this mechanism, antibodies can effectively "tag" a microbe or an infected cell for attack by other parts of the immune system, or can neutralize it directly.
A DNA vaccine is a type of vaccine that transfects a specific antigen-coding DNA sequence into the cells of an organism as a mechanism to induce an immune response.
Immunosuppressive drugs, also known as immunosuppressive agents, immunosuppressants and antirejection medications, are drugs that inhibit or prevent the activity of the immune system.
Haptens are small molecules that elicit an immune response only when attached to a large carrier such as a protein; the carrier may be one that also does not elicit an immune response by itself. The mechanisms of absence of immune response may vary and involve complex immunological interactions, but can include absent or insufficient co-stimulatory signals from antigen-presenting cells.
Hybridoma technology is a method for producing large numbers of identical antibodies, also called monoclonal antibodies. This process starts by injecting a mouse with an antigen that provokes an immune response. A type of white blood cell, the B cell, produces antibodies that bind to the injected antigen. These antibody producing B-cells are then harvested from the mouse and, in turn, fused with immortal myeloma cancer cells, to produce a hybrid cell line called a hybridoma, which has both the antibody-producing ability of the B-cell and the longevity and reproductivity of the myeloma.
The direct and indirect Coombs tests, also known as antiglobulin test (AGT), are blood tests used in immunohematology. The direct Coombs test detects antibodies that are stuck to the surface of the red blood cells. Since these antibodies sometimes destroy red blood cells they can cause anemia; this test can help clarify the condition. The indirect Coombs test detects antibodies that are floating freely in the blood. These antibodies could act against certain red blood cells; the test can be carried out to diagnose reactions to a blood transfusion.
An immunogen is any substance that generates B-cell (humoral/antibody) and/or T-cell (cellular) adaptive immune responses upon exposure to a host organism. Immunogens that generate antibodies are called antigens ("antibody-generating"). Immunogens that generate antibodies are directly bound by host antibodies and lead to the selective expansion of antigen-specific B-cells. Immunogens that generate T-cells are indirectly bound by host T-cells after processing and presentation by host antigen-presenting cells.
Immunogenicity is the ability of a foreign substance, such as an antigen, to provoke an immune response in the body of a human or other animal. It may be wanted or unwanted:
Artificial induction of immunity is immunization achieved by human efforts in preventive healthcare, as opposed to natural immunity as produced by organisms' immune systems. It makes people immune to specific diseases by means other than waiting for them to catch the disease. The purpose is to reduce the risk of death and suffering, that is, the disease burden, even when eradication of the disease is not possible. Vaccination is the chief type of such immunization, greatly reducing the burden of vaccine-preventable diseases.
Tuftsin is a tetrapeptide located in the Fc-domain of the heavy chain of immunoglobulin G. It has an immunostimulatory effect. It is named for Tufts University where it was first discovered in 1983.
Monoclonal antibodies (mAbs) have varied therapeutic uses. It is possible to create a mAb that binds specifically to almost any extracellular target, such as cell surface proteins and cytokines. They can be used to render their target ineffective, to induce a specific cell signal, to cause the immune system to attack specific cells, or to bring a drug to a specific cell type.
Polyclonal B cell response is a natural mode of immune response exhibited by the adaptive immune system of mammals. It ensures that a single antigen is recognized and attacked through its overlapping parts, called epitopes, by multiple clones of B cell.
Immunocontraception is the use of an animal's immune system to prevent it from fertilizing offspring. Contraceptives of this type are not currently approved for human use.
Monospecific antibodies are antibodies whose specificity to antigens is singular in any of several ways: antibodies that all have affinity for the same antigen; antibodies that are specific to one antigen or one epitope; or antibodies specific to one type of cell or tissue. Monoclonal antibodies are monospecific, but monospecific antibodies may also be produced by other means than producing them from a common germ cell. Regarding antibodies, monospecific and monovalent overlap in meaning; both can indicate specificity to one antigen, one epitope, or one cell type. However, antibodies that are monospecific to a certain tissue, or all monospecific to the same tissue because clones, can be polyvalent in their epitope binding.
Peptide-based synthetic vaccines are subunit vaccines made from peptides. The peptides mimic the epitopes of the antigen that triggers direct or potent immune responses. Peptide vaccines can not only induce protection against infectious pathogens and non-infectious diseases but also be utilized as therapeutic cancer vaccines, where peptides from tumor-associated antigens are used to induce an effective anti-tumor T-cell response.
Porcine epidemic diarrhea is a condition caused by the porcine epidemic diarrhea virus that leads to severe gastrointestinal disease in pigs.
Immunology is the study of the immune system during health and disease. Below is a list of immunology-related articles.
Passive antibody therapy, also called serum therapy, is a subtype of passive immunotherapy that administers antibodies to target and kill pathogens or cancer cells. It is designed to draw support from foreign antibodies that are donated from a person, extracted from animals, or made in the laboratory to elicit an immune response instead of relying on the innate immune system to fight disease. It has a long history from the 18th century for treating infectious diseases and is now a common cancer treatment. The mechanism of actions include: antagonistic and agonistic reaction, complement-dependent cytotoxicity (CDC), and antibody-dependent cellular cytotoxicity (ADCC).
Nicotine vaccine is a novel immunological strategy for treating nicotine addiction. Nicotine vaccine uses active immunization as the methodology to create polyclonal antibodies to the antigens, which is then used to treat drug abuse. The immune system is then able to identify nicotine as a foreign substance and initiate an immune reaction targeting the drug. As a result, the quantity of nicotine that enters the brain would decrease after receiving the vaccine. In preclinical studies, nicotine vaccines have demonstrated the ability to combat the negative effects of nicotine abuse, but none of the developed vaccines has been authorized for use in clinical trials as a smoking cessation strategy. Theoretically, the decrease of nicotine's rewarding effects should result in smoking cessation. Some companies have tested candidate vaccines in clinical trials, but evidence failed to show the adequate antibody responses or exhibit superior efficacy to factors concerning placebo.