The immune system is composed both functionally and anatomically of cellular and humoral compartments. The cellular component of immunity relies most heavily on lymphocytes dependent on the thymus for their development and maturation (T cells), while the humoral component consists of a class of soluble molecules found in the serum (antibodies) that are characterized by enormous diversity as well as specificity. These antibodies are synthesized by non-thymic cells derived from bone marrow precursors (B cells). Current research provides considerable evidence for the interdependence of the two arms of the immune response and the methods of modern immunology exploit an extensive understanding of both the cellular and humoral reactions. Although it is far beyond the scope of this book to convey even superficially the excitement that has permeated research into the cellular and molecular mechanisms that underlay the immune response, two books can be strongly recommended: one by Silverstein (1989) conveys the historical development of this field and the other by Paul (1993) presents its detailed scientific maturation.
B cell immunity, characterized most simply by the accumulation of specific antibodies to specific foreign molecules or antigens, has been the subject of intense biochemical and cellular study for almost a century. The early immunologists included Ehrlich, Pasteur, and Koch, who studied the resistance to infection produced by immunization with attenuated organisms and toxins. A critical conceptual advance was derived from the demonstration by Tiselius and Kabat (1938) that the antibody activity of serum resided in a particular electrophoretic fraction, the y-globulins. The past fifty years have witnessed the development of precise methods for the measurement of antibodies and their binding activity, an understanding of the molecular genetics of antibody molecules (which depends in large part on a set of novel reactions including the somatic rearrangement of immunoglobulin genes; Bernard et al., 1978; Seidman and Leder, 1978), the determination of the three-dimensional structure of a number of antibody fragments as well as of several antibody-antigen complexes (Davies et al., 1990), and the discovery and refinement of methods for isolating monoclonal antibodies (Köhler and Milstein, 1975; Gefter et al., 1977).
The practical advantage of an immunological approach to the study of proteins, carbohydrates, and cells is that, when properly exploited, the immune system can be cajoled into providing unique and powerful reagents. As with the preparation of most biological reagents, the production and purification of antibodies requires a clear need, a precise assay, careful consideration of the proper species in which the antibodies will be generated, and appropriate attention to detail throughout the many steps of these protocols. The nature and specificity of the reagents that can be generated, however, is limited only by the extent and clarity of one's imagination.
In this chapter, production of antibodies is presented. As with any biochemical reagent, simple and reliable assays are needed to detect their presence, amount, purity, and specificity. Although the radioimmunoassay as originally described by Yalow and Berson (1960) has played a major and critical role in the popularization of the use of immunologic
Induction of Immune
Contributed by David H. Margulies
Current Protocols in Immunology (2000) 2.0.1-2.0.3 Copyright © 2000 by John Wiley & Sons, Inc.
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