The Mouse in Science - Monoclonal Antibodies

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Title: The Mouse in Science - Monoclonal Antibodies

Author: Bennie I. Osburn, DVM, PhD, Dean
Donald J. Klingborg, DVM, Assistant Dean
Lynette A. Hart, PhD, Director
Mary W. Wood, Information Specialist
Amy Dassler, Editing and Graphic Design
Amy Mitchell, Research and Text

Publisher: School of Veterinary Medicine University of California

In 1975, Kohler and Milstein first fused lymphocytes to produce a cell line which was both immortal and a producer of specific antibodies. The two scientists were awarded the Nobel Prize for Medicine in 1984 for the development of this "hybridoma." The value of hybridomas to the field was not truly appreciated until about 1987, when MAbs were regularly produced in rodents for diagnostics.


What are Monoclonal Antibodies?

Monoclonal antibodies (MAbs) are:

* antibodies of exceptional purity and specificity

* components of the immune system

* able to recognize and bind to a specific antigen

Uses

Monoclonal antibodies are currently utilized in many diagnostic procedures, including:

* measuring protein and drug levels in serum

* typing tissue and blood

* identifying infectious agents

* identifying clusters of differentiation for the classification and follow-up therapy of leukemias and lymphomas

* identifying tumor antigens and auto-antibodies

* identifying the specific cells involved in the immune response

* identifying and quantifying hormones

Technology: The first step in the production of MAbs is to immunize a mouse with an antigen. When the mouse begins to produce antibodies to the antigen, its spleen is removed. Antibody-producing cells from the spleen are then fused with a myeloma cell line, one which is not Ab-producing and has been maintained in culture. The new fused cell line, which does produce antibodies, is grown briefly in culture and then re-injected into another mouse's peritoneum. Finally, the ascites fluid which contains monoclonal antibodies is harvested from the mouse.

Factors Affecting MAb Production
The purity of the antigen, the mouse strain which is used, the age and sex of the mouse, the tolerance of the individual mouse, and many other factors may affect the outcome of Ab-producing procedures. Because so many variables are present, one general way to maximize the animal's immune response is to use adjuvants. Adjuvants hold the antigen at the site of immunization and release it over a long period of time. Freund's Complete Adjuvant, which is more widely used than any other adjuvant, includes killed mycobacteria which serve to increase inflammation upon injection, and thereby enhance the mouse's immune response. This inflammation often results in painful lesions at the injection site.

ELISA
The Enzyme Linked ImmunoSorbent Assay (ELISA) was first developed by Engvall and Perlman in 1971. ELISA makes it possible to create a simple color test to detect and quantify the presence of an antigen, antibody, or hormone. This system is widely used because it can process large numbers of samples in a short time and can handle complex antigens, such as bacteria.

In this assay, the MAb is an antigen which has been anchored to a surface. This antibody then can be detected by a polyclonal antibody preparation which binds the monoclonal antibody. These polyclonal antibodies are linked to enzymes which generate a colored product in the presence of the MAb.

Commercial uses of the ELISA test are in high demand. Over-the-counter pregnancy tests make use of this principle by using MAbs directed to protein samples in urine. To monitor diabetes, glucose levels can be determined using a mediator molecule to interface biological and electronic components of a sensor which results in a current reading. Antibiotic residues in milk may be detected using an ELISA system which monitors the inhibition of a test organism's growth. ELISA is also used in food safety by indicating the presence of salmonella. This test is used to predict salmonella carrier status in cattle.

Human Monoclonal Antibodies
The production of MAbs has opened many doors in the area of immunotherapy. Human antibodies, however, are much more useful in this capacity than are those of mice, which may be rejected by the human immune system. Also, in some cases, MAbs from mice may elicit different responses than those from humans.

Human MAb production has proved to be quite complex. One current strategy involves transforming cells with Epstein-Barr virus and stabilizing them by extensive cloning. Another popular technique is the "humanization" of mouse MAbs. In this procedure, regions of the human myeloma protein are joined to the variable region of a mouse antibody. The production of true human antibodies is possible with the use of the Polymerase Chain Reaction, but limitations of this procedure are still in need of attention.

In Vitro Production.
The expansion of hybridomas in animals is becoming less acceptable due to humane and economic concerns. Several European countries have incorporated legislation limiting antibody production in mice. MAbs are extensively produced in vitro in Switzerland and Germany. Although in vivo production is relatively inexpensive, ascites fluid of mice may yield commercially unsuitable antibody. Two popular alternatives are bulk tissue culture in encapsulated or hollow fibre systems, and the expression of cloned antibody genes in high producing eukaryotes. The latter is accomplished through recombinant DNA technology.

Quality of Life for Mice: A Dilemma.
Freund's Complete Adjuvant (FCA) is the most effective adjuvant for antibody production in mice, but it creates inflammatory lesions at the inoculation site. The resulting discomfort to mice is a subject of widespread concern. When FCA is used, specific guidelines commonly require using smaller doses at each injection site and avoiding foot pad or intradermal injections. Due to the pain FCA causes, large-scale FCA use has been banned in the Netherlands and the United Kingdom. Alternative adjuvants are effective in certain situations.


GLOSSARY

adjuvant a substance that increases the antigenic response and is used to increase production of antibody.

antibody (Abs) a protein secreted by B lymphocytes in response to an antigen.

antigen a substance that induces the formation of antibodies.

ascites the accumulation of serum in the peritoneal cavity of the abdomen.

Freund's Complete Adjuvant (FCA) the adjuvant that produces very high antibody levels by stimulating a hypersensitive, painful inflammation at the injection site.

hybridoma the cell produced by the fusion of an antibody-producing cell and a multiple myeloma cell; this cell can produce a continuous supply of antibody.

lymphocyte a particular type of white blood corpuscle; B lymphocytes arise in bone marrow; lymphocytes in the thymus develop into T-cells.

monoclonal arising from a single cell, as in antibodies specific for a particular antigen and derived from hybridoma cells.

myeloma a tumor of B lymphocyte cells arising in the bone marrow.

(Mice in hands graphic)

The Mouse in Science is published by the UC Center for Animal Alternatives. The UCCAA mission is to disseminate information concerning animal alternatives so as to improve the well-being and quality of life of animals wherever possible, and to optimize their contribution to education and research.

Text copyrighted The Regents of the University of California 1996.