Polyclonal antibodies (pAbs) are
antibodies
An antibody (Ab), also known as an immunoglobulin (Ig), is a large, Y-shaped protein used by the immune system to identify and neutralize foreign objects such as pathogenic bacteria and viruses. The antibody recognizes a unique molecule of the ...
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
An antibody (Ab), also known as an immunoglobulin (Ig), is a large, Y-shaped protein used by the immune system to identify and neutralize foreign objects such as pathogenic bacteria and viruses. The antibody recognizes a unique molecule of the ...
molecules that react against a specific
antigen
In immunology, an antigen (Ag) is a molecule or molecular structure or any foreign particulate matter or a pollen grain that can bind to a specific antibody or T-cell receptor. The presence of antigens in the body may trigger an immune respons ...
, each identifying a different
epitope
An epitope, also known as antigenic determinant, is the part of an antigen that is recognized by the immune system, specifically by antibodies, B cells, or T cells. The epitope is the specific piece of the antigen to which an antibody binds. The p ...
.
Production
The general procedure to produce polyclonal antibodies is as follows:
#
Antigen
In immunology, an antigen (Ag) is a molecule or molecular structure or any foreign particulate matter or a pollen grain that can bind to a specific antibody or T-cell receptor. The presence of antigens in the body may trigger an immune respons ...
preparation
#
Adjuvant In pharmacology, an adjuvant is a drug or other substance, or a combination of substances, that is used to increase the efficacy or potency of certain drugs. Specifically, the term can refer to:
* Adjuvant therapy in cancer management
* Analgesi ...
selection and preparation
# Animal selection
# Injection process
# Blood serum extraction
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
Mammals () are a group of vertebrate animals constituting the class Mammalia (), characterized by the presence of mammary glands which in females produce milk for feeding (nursing) their young, a neocortex (a region of the brain), fur or ...
, 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
In immunology, an antigen (Ag) is a molecule or molecular structure or any foreign particulate matter or a pollen grain that can bind to a specific antibody or T-cell receptor. The presence of antigens in the body may trigger an immune respons ...
is injected into the mammal. This induces the B-
lymphocyte
A lymphocyte is a type of white blood cell (leukocyte) in the immune system of most vertebrates. Lymphocytes include natural killer cells (which function in cell-mediated, cytotoxic innate immunity), T cells (for cell-mediated, cytotoxic ad ...
s to produce
IgG
Immunoglobulin G (Ig G) is a type of antibody. Representing approximately 75% of serum antibodies in humans, IgG is the most common type of antibody found in blood circulation. IgG molecules are created and released by plasma B cells. Each IgG ...
immunoglobulin
An antibody (Ab), also known as an immunoglobulin (Ig), is a large, Y-shaped protein used by the immune system to identify and neutralize foreign objects such as pathogenic bacteria and viruses. The antibody recognizes a unique molecule of the ...
s specific for the antigen. This
polyclonal
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 ...
IgG
Immunoglobulin G (Ig G) is a type of antibody. Representing approximately 75% of serum antibodies in humans, IgG is the most common type of antibody found in blood circulation. IgG molecules are created and released by plasma B cells. Each 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 In pharmacology, an adjuvant is a drug or other substance, or a combination of substances, that is used to increase the efficacy or potency of certain drugs. Specifically, the term can refer to:
* Adjuvant therapy in cancer management
* Analgesi ...
(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
Titer (American English) or titre (British English) is a way of expressing concentration. Titer testing employs serial dilution to obtain approximate quantitative information from an analytical procedure that inherently only evaluates as positiv ...
, high affinity
antisera
Antiserum is a blood serum containing monoclonal or polyclonal antibodies that is used to spread passive immunity to many diseases via blood donation (plasmapheresis). For example, convalescent serum, passive antibody transfusion from a previ ...
for use in experimentation or diagnostic tests.
Adjuvants In pharmacology, an adjuvant is a drug or other substance, or a combination of substances, that is used to increase the efficacy#Pharmacology, efficacy or Potency (pharmacology), potency of certain drugs. Specifically, the term can refer to:
* Adju ...
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:
# surfactants which promote concentration of protein antigens molecules over a large surface area, and
# immunostimulatory molecules or properties. Adjuvants are generally used with soluble protein antigens to increase antibody titers and induce a prolonged response with accompanying memory.
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.
Animal selection
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:
# the amount of antibody needed,
# the relationship between the donor of the antigen and the recipient antibody producer (generally the more distant the phylogenetic relationship, the greater the potential for high titer antibody response) and
# the necessary characteristics
.g., class, subclass (isotype), complement fixing natureof the antibodies to be made. Immunization and phlebotomies are stress associated and, at least when using rabbits and rodents, specific pathogen free (SPF) animals are preferred. Use of such animals can dramatically reduce morbidity and mortality due to pathogenic organisms, especially ''Pasteurella multocida'' in rabbits.
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
Puberty is the process of physical changes through which a child's body matures into an adult body capable of sexual reproduction. It is initiated by hormonal signals from the brain to the gonads: the ovaries in a girl, the testes in a boy. ...
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.
Antigen preparation
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
A membrane transport protein (or simply transporter) is a membrane protein involved in the movement of ions, small molecules, and macromolecules, such as another protein, across a biological membrane. Transport proteins are integral transmembran ...
to increase immunogenicity and provide
T cell
A T cell is a type of lymphocyte. T cells are one of the important white blood cells of the immune system and play a central role in the adaptive immune response. T cells can be distinguished from other lymphocytes by the presence of a T-cell r ...
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
Keyhole limpet hemocyanin (KLH) is a large, multisubunit, oxygen-carrying, metalloprotein that is found in the hemolymph of the giant keyhole limpet, ''Megathura crenulata'', a species of keyhole limpet that lives off the coast of California, fro ...
(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.
Antigen quantity
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:
# rabbit, 50–1000 µg;
# mouse, 10–50 µg;
# guinea pig, 50–500 µg; and
# goat, 250–5000 µg.
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.
Peptide antibodies
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 it
ProtScalepage that require some degree of user knowledge to navigate. For a more simple peptide scoring tool there is
Antigen Profilertool 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:
* Extremely long repeats of the same amino acid (e.g. RRRR)
* Serine (S), Threonine (T), Alanine (A), and Valine (V) doublets
* Ending or starting a sequence with a proline (P)
* Glutamine (Q) or Asparagine (N) at the n-terminus
* Peptides over weighted with hydrophobic residues (e.g. V, A, L, I, etc....)
Reactivity
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
An emulsion is a mixture of two or more liquids that are normally immiscible (unmixable or unblendable) owing to liquid-liquid phase separation. Emulsions are part of a more general class of two-phase systems of matter called colloids. Althoug ...
adjuvant, retain greater native protein structure than those in water-in-oil emulsions.
Asepticity
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 0.22μm 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.
Adjuvants
There are many commercially available
immunologic adjuvant
In immunology, an adjuvant is a substance that increases or modulates the immune response to a vaccine. The word "adjuvant" comes from the Latin word ''adiuvare'', meaning to help or aid. "An immunologic adjuvant is defined as any substance that ...
s. 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.
Freund’s adjuvants
There are two basic types of
Freund's adjuvant
Freund's adjuvant is a solution of antigen emulsified in mineral oil and used as an immunopotentiator (booster). The complete form, Freund's Complete Adjuvant (FCA or CFA) is composed of inactivated and dried mycobacteria (usually '' M. tuberculo ...
s: Freund's Complete Adjuvant (FCA) and Freund's Incomplete Adjuvant (FIA). FCA is a water-in-oil emulsion that localizes antigen for release periods up to 6 months. It is formulated with mineral oil, the surfactant mannide monoleate and heat killed ''
Mycobacterium tuberculosis
''Mycobacterium tuberculosis'' (M. tb) is a species of pathogenic bacteria in the family Mycobacteriaceae and the causative agent of tuberculosis. First discovered in 1882 by Robert Koch, ''M. tuberculosis'' has an unusual, waxy coating on its c ...
'', ''
Mycobacterium butyricum'' or their extracts (for aggregation of macrophages at the inoculation site). This potent adjuvant stimulates both cell mediated and humoral immunity with preferential induction of antibody against epitopes of denatured proteins. Although FCA has historically been the most widely used adjuvant, it is one of the more toxic agents due to non-metabolizable mineral oil and it induces granulomatous reactions. Its use is limited to laboratory animals and it should be used only with weak antigens. It should not be used more than once in a single animal since multiple FCA inoculations can cause severe systemic reactions and decreased immune responses. Freund's Incomplete Adjuvant has the same formulation as FCA but does not contain mycobacterium or its components. FIA usually is limited to booster doses of antigen since it normally much less effective than FCA for primary antibody induction. Freund's adjuvants are normally mixed with equal parts of antigen preparations to form stable emulsions.
Ribi Adjuvant System
Ribi adjuvants are oil-in-water emulsions where antigens are mixed with small volumes of a metabolizable oil (squalene) which are then emulsified with saline containing the surfactant Polysorbate 80. This system also contains refined mycobacterial products (cord factor, cell wall skeleton) as immunostimulants and bacterial monophosphoryl lipid A. Three different species oriented formulations of the adjuvant system are available. These adjuvants interact with membranes of immune cells resulting in cytokine induction, which enhances antigen uptake, processing and presentation. This adjuvant system is much less toxic and less potent than FCA but generally induces satisfactory amounts of high avidity antibodies against protein antigens.
Titermax
Titermax represents a newer generation of adjuvants that are less toxic and contain no biologically derived materials. It is based upon mixtures of surfactant acting, linear, blocks or chains of nonionic copolymers polyoxypropylene (POP) and polyoxyethylene (POE). These copolymers are less toxic than many other surfactant materials and have potent adjuvant properties which favor chemotaxis, complement activation and antibody production. Titermax adjuvant forms a microparticulate water-in-oil emulsion with a copolymer and metabolizable squalene oil. The copolymer is coated with emulsion stabilizing silica particles which allows for incorporation of large amounts of a wide variety of antigenic materials. The adjuvant active copolymer forms hydrophilic surfaces, which activate complement, immune cells and increased expression of class II major histocompatibility molecules on macrophages. Titermax presents antigen in a highly concentrated form to the immune system, which often results in antibody titers comparable to or higher than FCA.
Specol:
Specol is a water in oil
adjuvant In pharmacology, an adjuvant is a drug or other substance, or a combination of substances, that is used to increase the efficacy or potency of certain drugs. Specifically, the term can refer to:
* Adjuvant therapy in cancer management
* Analgesi ...
made of purified
mineral oil
Mineral oil is any of various colorless, odorless, light mixtures of higher alkanes from a mineral source, particularly a distillate of petroleum, as distinct from usually edible vegetable oils.
The name 'mineral oil' by itself is imprecise, ...
. It has been reported to induce
immune response
An immune response is a reaction which occurs within an organism for the purpose of defending against foreign invaders. These invaders include a wide variety of different microorganisms including viruses, bacteria, parasites, and fungi which could ...
comparable to
Freund's adjuvant
Freund's adjuvant is a solution of antigen emulsified in mineral oil and used as an immunopotentiator (booster). The complete form, Freund's Complete Adjuvant (FCA or CFA) is composed of inactivated and dried mycobacteria (usually '' M. tuberculo ...
in rabbit and other research animal while producing fewer histological lesions.
Pharmaceutical uses
Digoxin Immune Fab is the antigen binding fragment of polyclonal antibodies raised to
Digitalis
''Digitalis'' ( or ) is a genus of about 20 species of herbaceous perennial plants, shrubs, and biennials, commonly called foxgloves.
''Digitalis'' is native to Europe, western Asia, and northwestern Africa. The flowers are tubular in sha ...
derivative as a hapten bound to a protein and is used for the reversal of life-threatening
digoxin or
digitoxin
Digitoxin is a cardiac glycoside used for the treatment of heart failure and certain kinds of heart arrhythmia. It is a phytosteroid and is similar in structure and effects to digoxin, though the effects are longer-lasting. Unlike digoxin, w ...
toxicity.
Rho(D) immune globulin
Rho(D) immune globulin (RhIG) is a medication used to prevent RhD isoimmunization in mothers who are RhD negative and to treat idiopathic thrombocytopenic purpura (ITP) in people who are Rh positive. It is often given both during and followin ...
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
Hemolytic disease of the newborn, also known as hemolytic disease of the fetus and newborn, HDN, HDFN, or erythroblastosis foetalis, is an alloimmune condition that develops in a fetus at or around birth, when the IgG molecules (one of the five ...
.
Rozrolimupab
Rozrolimupab is a recombinant antibody mixture, specifically containing 25 Rhesus D, or RhD, human antibodies, and is currently under development by Symphogen for the treatment of immune thrombocytopenic purpura (ITP) and for the prevention of ...
is the anti-
RhD recombinant human polyclonal antibody composed of 25 unique
IgG1
Immunoglobulin G (Ig G) is a type of antibody. Representing approximately 75% of serum antibodies in humans, IgG is the most common type of antibody found in blood circulation. IgG molecules are created and released by plasma B cells. Each IgG a ...
antibodies and is used for the treatment of immune
thrombocytopenia purpura and prevention of isoimmunization in
Rh-negative
The Rh blood group system is a human blood group system. It contains proteins on the surface of red blood cells. After the ABO blood group system, it is the most likely to be involved in transfusion reactions. The Rh blood group system consists ...
pregnant women.
REGN-COV2 (
Regeneron Pharmaceuticals
Regeneron Pharmaceuticals, Inc. is an American biotechnology company headquartered in Westchester County, New York. The company was founded in 1988. Originally focused on neurotrophic factors and their regenerative capabilities, giving rise to ...
) – potential treatment for people with
COVID-19
Coronavirus disease 2019 (COVID-19) is a contagious disease caused by a virus, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The first known case was COVID-19 pandemic in Hubei, identified in Wuhan, China, in December ...
and to prevent
SARS-CoV-2
Severe acute respiratory syndrome coronavirus 2 (SARS‑CoV‑2) is a strain of coronavirus that causes COVID-19 (coronavirus disease 2019), the respiratory illness responsible for the ongoing COVID-19 pandemic. The virus previously had a ...
coronavirus infection.
Advantages
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.
See also
*
Plasma B cell
Plasma cells, also called plasma B cells or effector B cells, are white blood cells that originate in the lymphoid organs as B lymphocytes and secrete large quantities of proteins called antibodies in response to being presented specific substa ...
*
Clone (B-cell biology)
The process of immunological B-cell maturation involves transformation from an undifferentiated B cell to one that secretes antibodies with particular specificity. This differentiation and activation of the B cell occurs most rapidly after exposur ...
*
Polyclonal response
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 ...
References
External links
Nature News piece on polyclonal antibody therapyImmunological Measures of Antigenicity (online textbook, see section 11.1.)Polyclonal antibody in Dictionary of Cell and Molecular Biology*
Generating antibodies at openwetware (technical description for researchers)
Polyclonal vs Monoclonal Antibodies
{{immune_system
Immunology
Immune system
Biotechnology
Glycoproteins
Reagents for biochemistry