Production
These antibodies are typically produced by immunization of 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 polyclonal 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:
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.
[edit] 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 [e.g., class, subclass (isotype), complement fixing nature] of 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 5.5-6.5lbs) 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.
These antibodies are typically produced by immunization of 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 polyclonal 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:
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.
[edit] 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 [e.g., class, subclass (isotype), complement fixing nature] of 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 5.5-6.5lbs) 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.
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