Vertebrate immune systems are capable of producing antibodies to a greater or lesser degree in response to the presence of a foreign protein within the tissues of the animal. The presence of the foreign protein initiates a sequence of events, mediated by the cells of the immune system that lead to the release of antibody molecules in blood and some body secretions.

Antibodies produced by vertebrate immune systems bind strongly to the protein that elicited their formation, and it is this unique ability that is harnessed in all branches of immunochemistry.

From: Methods in Molecular Biology, vol. 295: Immunochemical Protocols, Third Edition. Edited by: R. Burns © Humana Press Inc., Totowa, NJ

Vertebrates have evolved this immunological strategy to help them combat pathogens of viral, bacterial, and fungal origin; however, almost all foreign substances, regardless of source, can induce antibody responses. The immunological response in mammals is particularly well developed, and it is this group of vertebrates that are normally used for antibody production.

There are two main approaches for antibody production in vertebrates, each having strengths and weaknesses depending on intended application. Before discussing these two approaches, it would be beneficial to describe some background immunology.

Mammalian embryos are extremely tolerant of foreign proteins while still in utero, and all substances within the developing organism are accepted as "self." This is essential during development to ensure that immune responses are not raised to proteins and pep-tides produced during this time. Any immunological response to developmental proteins, hormones and growth factors would have disastrous results.

Shortly before or immediately after birth, the neonatal immune system matures and learns to differentiate between "self" and "non-self" (1). The immature immune system contains millions of cells within the bone marrow capable of producing antibodies (B-lym-phocytes). This cell population is in effect a "starter pack," containing cells that will be capable of responding to a huge number of target proteins. These neonatal lymphocytes are produced by random reassortment of the antibody genes and because of this many of them will recognize and respond to proteins within the developing individual. A process of clonal deletion takes place and any lymphocytes, which recognize proteins within the developing organism, are killed. As the young mammal matures, it is incapable of mounting an immunological response to "self" antigens as a result of the process of clonal deletion.

Remaining B-lymphocytes in the bone marrow have the potential to respond to an enormous number of foreign substances (antigens). Once exposed to antigens, the cells, which have the best fit antibody to the target, undergo clonal expansion to increase the cell numbers and affinity maturation to increase the specificity (fit) of the antibody molecules produced.

The ability of the mammalian immune system to respond to foreign substances is based on the molecular shape of antigen fragments produced as a result of digestion by cells called macrophages. The antigen fragments produced by this process are generally about the size that one antibody-binding site can physically adhere to. These fragments are known as epitopes, and although there will be many on any target substance, a single antibody will only be able to recognize and bind to one of them.

The response to any antigen will involve the recruitment of many B-lymphocytes, each making antibodies to an individual epitope on the target molecule. These lymphocytes, which have responded to the epitopes on the antigen, undergo clonal expansion so that many descendant B-lymphocytes are produced from each of the original cells. This process gives rise to a population of cells descended from single progenitors (clones) each with their own specific antibody to epitopes on the antigen. These clones of cells are resident in lym-phoid tissue and are particularly concentrated in the marrow, spleen, and gut-associated lymphoid tissue. The resulting pool of antibody molecules produced by these cells is known as polyclonal antibody, because it is derived from multiple clones, each with unique specificity to single epitopes.

Monoclonal antibodies are produced as a result of immortalizing and expanding the individual antibody secreting cells artificially in tissue culture (2). Cells grown in this way all have identical epitope specificity and because they are derived from single clones, their product is known as monoclonal antibody. Cells that secrete monoclonal antibodies are known as hybridomas and are typically derived by fusion of two cell types. B-lymphocytes, which have the capacity to make antibody, are obtained from a donor spleen and are physically fused to a tumor cell line, which is immortal. The resulting hybridom as are immortal and produce antibody into the synthetic medium in which they are growing.

Exposure to antigens can be by a variety of routes but ultimately the cellular changes leading to B-lymphocyte activation are blood borne. Natural immunization takes place as a consequence of infection through respiratory, digestive, urogenital and skin surfaces. Medical immunization to prevent infection by pathogens is normally conducted by intramuscular injection, although other routes, such as oral dosing for poliomyelitis and intradermal injection for tuberculosis, may be used. The main feature, which characterizes immunization, is the presentation of antigens to the cells of the immune system, which induces B-lymphocyte priming. These primed B-cells will undergo clonal expansion and will secrete antibody until the antigen has been destroyed. As soon as the antigen has been removed, the B-cell lineage making antibodies to it will become quiescent and will form a stable population within the tissues of the immune system (memory cells). If the antigen is encountered again by the organism these quiescent B-cells can undergo rapid clonal expansion and can mount an antibody response much faster than during the primary challenge. Each time that the immune B-cells are exposed to the antigen, the affinity (fit) of the antibody produced will be improved and the number of quiescent B-cells after each challenge increases. Each challenge also increases the amount of antibody produced in the blood and after three or four immunizations the individual reaches a status of hyperimmunity. This is characterized by high levels of circulating specific antibody typically in the range of 10-20 mg/mL of serum. Hyperimmunity is rarely ever seen as a result of natural immunization but is commonly used for the in vivo production of polyclonal antibody. Risks are associated with hyperimmunity as further exposure to the antigen can lead to an overwhelming immune response known as anaphylaxis, which can be rapidly fatal. Paradoxically, repeated exposure to the antigen can lead to immunological tolerance, where the B-cells making the antibody are destroyed by the immune system leaving the individual unable to mount an immune response to the antigen.

As previously stated, immunization is a phenomenon mediated by cells of the immune system and normally is the result of a blood-borne challenge by antigen. The route of introduction can be very important in determining how well the individual will respond to an antigen.

It is extremely important when immunizing animals for antibody work to choose the correct approach for the type of antigen to be used. This chapter describes a number of immunization routes for polyclonal antibody production, monoclonal antibody cell donors and also one method for inducing selective immune tolerance as a preparative method.

Polyclonal and monoclonal antibodies should be seen as complimentary in their use. Each has strengths and weaknesses and the choice of which to use should be carefully evaluated before embarking on antibody production. In general, polyclonal antibodies have a much broader specificity because the antiserum pool comprises many species of antibody molecule each with different target epitopes on the antigen. This lack of specificity can be advantageous in situations where variation in the target substance is known and polyclonal antibodies may provide a more robust test. Monoclonal antibodies are derived from clonal cell lines and their specificity is directed to a single epitope on the antigen. The highly specific nature of the monoclonal antibody allows the development of assays where two very closely related substances can be differentiated from each other. Examples of these highly specific tests are found in virus testing for strain differentiation and in clinical assays where levels of a synthetic hormone may be detected in spite of the presence of its naturally occurring counterpart.

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