Immunodiffusion

Alfred J. Crowle

• Publisher: Academic Press
• Year: 2014
• Print ISBN: 9780121981563
• eBook ISBN: 9781483267272
• Edition: 2
• Topic: Medicine
• Subtopic: Diseases & Allergies

Cancer and Chemotherapy, Volume III: Antineoplastic Agents is a collection of articles that deals with the treatment of cancer using drugs. The collection describes the various drugs that are used, the therapeutic approaches being taken, and agents that are being developed. Part I is a general review of anti-cancer drugs as regards their action mechanisms, pharmacokinetics, pharmacology, known toxicities, and clinical utility. These drugs include alkylating agents such as mitomycin C and nitrosoureas; plant alkaloids such as maytansine; antibiotics such as anthracyclines; platinum-containing complexes; antimetabolites; and hormones. Part II examines the molecular pharmacology of some major drug classes, namely, bleomycin and anthracycline. The text also discusses the chemistry, mechanism, and any structure-activity relationships found in these drug classes. Part III discusses in detail the clinical pharmacology of some antitumor drugs, for example, cisplatin and nitrosoureas. The text includes the clinical applications, biochemistry, metabolism, and the use of mathematical models in interpreting or describing resulting data. The book is helpful for pharmacologists, molecular biologists, and scientists involved in cancer-research.

Chapter 1

Basic Information

The purposes of this chapter are to introduce the reader to the two principal reagents of immunodiffusion tests, antiserum and antigen, and to explain the basic physical events and processes of these tests so as to prepare him for using and understanding immunodiffusion techniques.

Antiserum

Antiserum is the fundamental reagent of immunodiffusion and provides its great versatility and high specificity. It is used to detect, characterize, and quantitate antigens. Antiserum is produced by animals exposed appropriately to antigen (Weiser et al., 1969). An animal’s lymphoid system, recognizing antigen as a foreign substance and an implied biological threat (e.g., infection, tumor), manufactures proteins that react selectively with this foreign “body” and therefore are known as “antibodies.” The antibodies used in immunodiffusion tests are most commonly obtained by drawing blood from the antigen-immunized animal, allowing the blood to clot, centrifuging out the clot and accompanying blood cells, and drawing off the clear, amber-colored supernatant antiserum.

Antibodies

Serum contains many kinds of dissolved macromolecules, most of them proteins. These are classified electrophoretically into major groups by increasing isoelectric point (i.e., decreasing electrophoretic mobility) as prealbumins, albumin, α-globulins, β-globulins, γ-globulins, and basic proteins. In turn, these are subclassified by function, molecular size, solubility, and antigenic composition (Weiser et al., 1969). Most antiserum antibodies are γ-globulins termed, collectively, “immunoglobulins” because of their functions, and referred to by formula as “Ig” with a following letter to indicate class (e.g., IgA: Weiser et al., 1969; Smith, 1966). Antiserum contains several different classes of immunoglobulin which may or may not be antibodies against the immunizing antigen. Those that are antibodies usually are heterogeneous, differing in how they react with the same antigen and in what effect they consequently produce.

Solubility in distilled water distinguishes between two major varieties of immunoglobulin. Those precipitating when dialyzed against distilled water are euglobulins; those remaining in solution are Pseudoglobulins (Boyd, 1966). Against protein antigens, rabbits produce primarily euglobulins, whereas horses form principally Pseudoglobulins. But each species of animal also can, and usually does, produce small amounts of the other type of antibody (Siskind, 1966; Johnston and Allen, 1968). The principal euglobulin antibodies are electrophoretically classed as γ₂- or Ig₂-globulins because they are more cathodic than the pseudoglobulins, which, correspondingly, are γ₁-globulins. Though obsolescent, the terms euglobulin and Pseudoglobulin remain useful for indicating whether or not antibodies can be used in distilled water, which may be important in some immunodiffusion tests.

Different classes of antibody molecules share many chemical, physical, and biological characteristics. But because of differences in amino acid constitution they can be distinguished from each other immunologically (Abramoff and La Via, 1970). For instance, rabbit antiserum specific for one class of human immunoglobulin will not cross-react with another class of human immunoglobulin. By this and associated criteria, characterized human immunoglobulins have been classified as IgG, IgM, IgA, IgD, and IgE (Abramoff and La Via, 1970). Such nomenclatural systematization for immunoglobulins is relatively recent. Although it is being applied to antisera of lower animals as quickly as data accumulate and are interpreted, most classes of lower animal immunoglobulins have not yet been identified with their human serum counterparts. Consequently, other interim designations for antibody classes which only suggest similarities to human serum immunoglobulins are frequently used. For example, a 19 S animal immunoglobulin may be called γ_1M because it is a macromolecular γ-globulin with a γ₁ mobility in the immunoelectrophoretic pattern for that animal’s serum. But it should not be called IgM without considerable proof of its homology with human serum IgM.

The term “19 S” above refers to the physical characteristic of molecular size as estimated by ultracentrifugal sedimentation, in which “S” signifies “Svedberg” (Boyd, 1966). The class of antibody most frequently used in immunodiffusion tests is an antigen-precipitating 7 S immunoglobulin (precipitin) of molecular weight approximately 175,000 (Tran Van Ky et al., 1966a; Remington et al., 1962). Other classes of antibody may be larger because of attached accessory structures (e.g., 11 S secretory IgA in man, of molecular weight 400,000; Dayton et al., 1971), because they polymerize (e.g., 11 S and 14 S chicken precipitins: Kubo and Benedict, 1969; Van Orden and Treffers, 1968a; Hersh and Benedict, 1966), or because they are manufactured by the body in pentamers (e.g., 19 S IgM antibodies of molecular weight 900,000: Smith, 1966; Wahl et al., 1965; Abramoff and La Via, 1970). Occasionally, biologically active pieces of antibody also may be encountered—for instance, in urine (Remington et al., 1962) or in cattle antiserum (Cowan, 1966b). For immunodiffusion these molecular size distinctions are important, both because of correspondingly different rates of antibody diffusion and because of associated contrasts in reactions with antigen (Paul and Benacerraf, 1966). In recent years the ultracentrifuge has given way to simpler, less expensive ways of estimating antibody molecular size, such as measuring absolute or relative rates of diffusion through agar gels, determining diffusion-limiting pore size in semisolid media, or measuring gel-filtration R_f values (see Chapters 6 and 7).

By definition, all antibodies must be able to complex specifically with antigen. But antibodies differ in effects produced by such complexing and in conditions required for development of these effects. Indeed, they are known by their effects as precipitins (precipitate dissolved antigens), agglutinins (aggregate and sediment suspended antigens), complement-fixing antibodies (on combining with antigen they fix and activate enzymatic serum proteins known collectively as “complement”), opsonins (they combine with particulate antigens to facilitate their phagocytosis), and blocking antibodies (they interfere with manifestations of other kinds of antibodies). Antibody activities need not correspond with antibody immunoglobulin classification, since different classes of antibody may produce similar reactions with antigen. For instance, both γ_M- and γ_G-globulins in an antiserum can be precipitins (Pike, 1967; Tran Van Ky et al., 1966a). On the other hand, a given antiserum will be likely to contain various antibodies with differences in both effect on and avidity for the same antigen (Carter and Harris, 1967; Boyd, 1966; Abramoff and La Via, 1970), and among these, individual antibodies will differ as to the portions (determinants) of the antigen with which they combine (Weiser et al., 1969). These different kinds of antibody in a single antiserum, with their individual variations in relation to one antigen, and their competitive (Fiset, 1962; Christian, 1970) or complementary (Carter and Harris, 1967; Moore, 1961) interplay with each other through combination with the same antigen, define the overall antibody activity of the antiserum (Klinman et al., 1966). As will be seen below, this total activity also is affected importantly by other nonantibody constituents of the antiserum.

Immunodiffusion tests most commonly use precipitins. But in some, antibody complexed with antigen forms clear or “negative” precipitin bands in agar gels, instead of opaque ones (Moore, 1961; Silverstein et al., 1958); and there are immunodiffusion tests that detect blocking antibodies (Patterson et al., 1964a), complement-fixing antibodies (Milgrom and Loza, 1966; Paul and Benacerraf, 1966), agglutinins (Milgrom and Loza, 1967), and antibodies that form no more than primary complexes with antigen (Freeman and Stavitsky, 1966; see Chapter 7 for additional examples). The following discussion centers on precipitins because of their primacy in immunodiffusion. The characteristics and uses of nonprecipitating antibodies in this technique will become evident partly as a by-product of this discussion and partly with later description of specific tests using these antibodies.

PRECIPITINS

Precipitins are antibodies that insolubilize antigen; hence, an antiserum that produces a precipitate when mixed with antigen solution contains precipitins. But this precipitating capacity for an antiserum is the product of complex agents and events including nature of antibodies, interaction between antibodies, interplay with nonantibody serum constituents, physicochemical conditions, and nature of antigen. Consequently, only a functional definition of precipitins is possible, although most frequently these antibodies are 7 S γ-globulins that are divalent and have a high affinity for antigen.

Precipitins can be 30 S (Cowan, 1966b), 19 S (Josephson et al., 1962; Cowan and Trautman, 1965; Pike, 1967), 14 S (Orlans et al., 1961), 7 S (Siskind, 1966), or even 4.5 S globulins (Cowan, 1966b). Their electrophoretic mobility depends on the species of animal making them, on the antigen inducing them, and on the immunization protocol employed (Christian, 1970). Rabbits tend to make γ₂-globulin precipitins (Siskind, 1966); horses more copiously make γ₁-globulin precipitins (Johnston and Allen, 1968); precipitins frequently occur in both electrophoretic classes of globulin in guinea pigs (Wilkerson and White, 1966) and mice (Krøll, 1970) and occasionally also in man and monkeys (Hillyer, 1969). Guinea pigs injected with foot-and-mouth disease virus produced, within 4 days, 19 S γ₁-globulin precipitins which could neutralize virus but not fix complement; but after 15 days they had ceased production of this antibody and instead were manufacturing 7 S γ₂-globulin precipitins which could both fix complement and neutralize virus (Cowan and Trautman, 1965; Graves et al., 1964). Seven days after infection with the same virus, cattle were making precipitins of 19 S and 30 S γ₁-globulin, but later they made predominantly 7 S and 4.5 S γ₁- and γ₂-globulin precipitins (Cowan, 1966b).

Precipitins are called R- or H-type according to how they precipitate antigens (see section on antigen–antibody precipitation, below); and they can be either pseudo- or euglobulins. But production of one or the other of these types is not an exclusive characteristic of just certain species of animals. For example, conventionally immunized rabbits produce pseudo- and euglobulins, and bo...

Table of contents

• Cover image
• Title page
• Table of Contents
• Copyright
• Dedication
• Preface to the First Edition
• Introduction
• Chapter 1: Basic Information
• Chapter 2: General Information
• Chapter 3: Single Diffusion Tests
• Chapter 4: Double Diffusion Tests
• Chapter 5: Immunoelectrophoresis
• Chapter 6: Electroimmunodiffusion
• Chapter 7: Ancillary Immunodiffusion Techniques
• Chapter 8: History
• References
• Glossary
• Adjuvants
• Selected Immunodiffusion Electrolyte Solutions (All formulas are made up to 1 liter)
• Selected Immunoelectrophoresis and Electroimmunodiffusion Buffers (All formulas are made up to 1 liter)
• Immunochromatography Buffers (Both formulas made up to 1 liter)
• Selected Protein Stains
• Selected Lipid Stains
• Selected Polysaccharide Stains
• Selected Nucleic Acid Stains
• Selected Double Stains
• Subject Index