Antibody Techniques

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PREFACE

This manual is intended to serve as a guide for those who have little or no practical laboratory experience in techniques using antibodies. The primary purpose is to provide a step-by-step, bench-top reference detailing the protocols and recipes of common laboratory procedures that use antibodies as reagents for research in all areas of biological science. Concurrently, an effort has been made, where appropriate, to provide a theoretical background to the procedures described. This book is not intended to provide a survey of all methods and techniques available because readers are probably better served by a critical evaluation of a number of procedures rather than a mere description of all of them. We hope that this book will provide, in one volume, a common consensus of antibody-related protocols currently in practice in research and clinical biological sciences.

The first half of this book encompasses subjects relevant to how antibodies are produced in research laboratories. The introductory chapter provides a theoretical overview of how the immune system works, particularly regarding antigen processing and presentation to T and B cells, and subsequent immunoglobulin gene expression. Chapters 2 through 7 furnish procedures for production of polyclonal, monoclonal, and recombinant antibodies; proper methods for use and care of laboratory animals; recent developments in adjuvant research; and large-scale production and purification of antibodies. The second half of the book (Chapters 8 through 15) describes particular techniques that use antibodies as immunoreagents in both research and clinical settings. These include the use of antibody-binding bacterial proteins, immunoaffinity purification of antigens, preparation of antibody heteroconjugates, immunostaining, cell sorting, immunoassays, and immunoscreening of expression libraries.

As the editors, we extend our appreciation to the individual contributors who have provided their expertise, time, and effort on this project. Finally, this guide could not have been written without the encouragement and guidance of Dr. Lorraine Lica and the editorial staff at Academic Press.

ERIK P. LILLEHOJ

VEDPAL S. MALIK

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ANTIBODY MOLECULES AND THE IMMUNE RESPONSE: THEORETICAL BACKGROUND

IAN M. ZITRON

Publisher Summary

The normal function of the immune system is to provide protection against invasion by pathogens. It is, unfortunately, also the source of less welcome events, such as graft rejection and autoimmune diseases, in which the system reacts against normal body constituents. An immune response, whether it be beneficial or harmful, is a multi-layered phenomenon. There are two forms of immunities present in a living body—innate immunity that is present through the course of the being’s lifetime and acquired immunity that stems from adaptations by the body to foreign stimulus with effector functions working in response to that particular stimulus. Adaptation shows itself in two characteristics, specificity and memory, that combine to give the system its efficiency. The three defining characteristics of the acquired response—specificity, memory, and self/nonself discrimination—reflect the properties and activities of lymphocytes. These cells are directly responsible for the acquired immune response, although in many instances the response also requires the involvement of cells of the monocyte/macrophage lineage.

I THE ORGANIZATION OF IMMUNE PROTECTION

The normal function of the immune system is to provide protection against invasion by pathogens. It is, unfortunately, also the source of less welcome events, such as graft rejection and autoimmune diseases, in which the system reacts against normal body constituents. An immune response, whether it be beneficial or harmful, is a multi-layered phenomenon. The purpose of this chapter is twofold. The first is to provide an introductory description of the various layers, in conjunction with an indication of some of the interactions that occur both between the layers and within them. The second is a more detailed consideration of those aspects of the system that are of most interest and importance to scientists who wish simply to use immunological techniques, particularly antibodies, in their research. The emphasis is on the genetic, cellular, and molecular events that give rise to the production of antibodies, rather than on immunochemistry per se. Nonimmunologists frequently complain that the language and terminology used in immunology present major barriers to an appreciation of the system, obscuring rather than clarifying. Although this chapter is in no sense a replacement for a comprehensive dictionary, it may make some aspects of the immune system more understandable.

II INNATE AND ACQUIRED IMMUNITY

The first broad separation is the discrimination between innate and acquired immunity. Innate immunity is, as its name suggests, present at birth and persists throughout life. It represents the first line of defense against insult (the word insult is used broadly in immunology and refers to invasion by pathogenic microorganisms, parasites, and, in some instances, tumors) and is composed of a number of physical, cellular, and chemical barriers. Skin and mucous membranes physically impede invasion. Chemical barriers include gastric pH, enzymes such as lysozyme in tears and saliva, and other biologically active molecules such as the interferons and the proteins of the complement system. Cells, such as polymorphonuclear leukocytes (PMNs) and natural killers (NKs) are also integral components of this aspect of protection. Thus at least three layers of protection exist in innate immunity. Although each is mechanistically quite different in the way in which protection is effected, there are characteristics that all layers have in common. Each layer is either continuously present, or very rapid in response to insult. The layers show no specificity vis a vis the insult: they are incapable of discriminating between different pathogens. Also, they show no memory of prior insults: neither the quality nor the quantity of innate immunity is increased by a second exposure to the same pathogen. Acquired immunity differs from innate immunity in each of these characteristics. Table I shows a comparison between innate and acquired immunity. The elements of innate immunity provide a significant degree of protection; the severity of immune deficiency syndromes that reflect a lack of individual components of this system provides ample evidence of its importance.

TABLE I

Differences between Innate and Acquired Immunity

In contrast, acquired immunity is adaptive, since it responds to the particular insult with effector functions that act only on that insult. Adaptation shows itself in two characteristics, specificity and memory, which combine to give the system its efficiency. Figure 1 shows the time courses and magnitudes of (antibody) responses to two immunologically unrelated substances, designated A and B. The figure illustrates the difference in response subsequent to first (primary) and second (secondary) exposure to a foreign substance; this behavior reflects immunological memory. Specificity is the ability to discriminate between different insults; in the figure, the primary immunization with A elicits antibodies that recognize A only; the memory that this response generates is specific for A. Specificity may be maintained even when the substances are closely related biologically and chemically. One of the best known examples of the system’s ability to distinguish subtle differences is the human ABO blood group system (Landsteiner, 1946). Perhaps an even more striking example of the level of discrimination possible is that antibody populations may be identified that are capable of discriminating between closely related nitrophenyl groups (Little and Eisen, 1969). Immunological memory is very much like that observed in the functioning of the nervous system: the cells have the ability to recognize, and respond to, the second (or third, and so on) exposure to a given insult in a way that is both quantitatively and qualitatively different from the response to the first, or primary, exposure. Moreover, memory is itself specific because the enhanced response subsequent to re-exposure is directed only toward the foreign substance used for initial immunization.

FIGURE 1 Kinetic and quantitative differences between primary and secondary humoral (antibody) responses. The primary response to immunization with A shows a lag period of ~7 days, followed by a logarithmic increase, a relatively low plateau titer, and a rapid decay. In contrast, secondary immunization with A gives rise to a response with a shorter lag period, a steeper logarithmic increase reaching a significantly higher plateau (note the logarithmic scale of the ordinate), and a slow decay phase. The difference between primary and secondary responses reflects immunological memory, which has been generated as a consequence of primary immunization, but which requires rechallenge to be expressed. The secondary immunization with A is accompanied by primary immunization with an immunologically distinct substance, B. The indicated response to B is characteristic of a primary response, indicating that memory itself is specific.

The acquired immune response shows an additional characteristic that is of considerable significance for homeostasis, the ability to discriminate between self and nonself. The phenomenon was first shown in the 19th century by Paul Ehrlich (1900), who gave it the name Horror Autotoxicus. In its simplest terms, the observation is that, although vertebrates may be immunized with foreign (i.e., nonself) material and be shown to mount an immune response, spontaneous responses to the animal’s own molecules leading to autoimmune disease (i.e., anti-self) are rare. The process by which recognition of self is minimized is referred to as self-tolerance. Self-tolerance is not simply a failure to recognize self components, but is an active process involving the regulation of lymphocyte survival and function (see subsequent text and Figures 7 and 8; Nossal, 1994).

FIGURE 7 Developmental sequence of B cells. B cells are derived from the pluripotential stem cell, which has self-renewing capacity and is also able to give rise to all elements of the hemopoietic system. Interleukin-3 (IL-3) is required for development of the lymphoid progenitor, a cell that is irreversibly committed to the lymphocytic pathway. This cell can give rise to both B and T lymphocytes. The latter require migration from bone marrow to thymus for development. Pro-B cell development is driven by IL-7. Pro-B cells express CD45 and surrogate light chain and are the first unambiguous step in the B cell pathway. Pre-B cells are the stage at which class II MHC becomes expressed and the Ig loci undergo the rearrangements that result in the expression of variable region domains. The heavy chain locus rearranges prior to the light chain. Immature B cells express mIgM and the signal-transducing complex of Ig-α and Ig-β. As such, these are the first cells in the pathway to be responsive to external immunogen. However, exposure to immunogen results in tolerization. The final step is the mature B cell. This is recognized by the co-expression of mIgM and mIgD. These cells migrate to the secondary lymphoid organs. They respond to immunogen by activation into the G1 phase of the cell cycle, the first step leading to a humoral response.

FIGURE 8 Intrathymic development of α/β T cells. T cell precursors from the bone marrow enter the thymic cortex; these cells do not express the T cell markers CD3,4/8 or T cell receptor (TCR) and are referred to as triple-negative (TN) cells. TCRβ rearrangement permits the expression of the β chain, which is expressed on the cell surface with a surrogate α chain, gp33. The cells then acquire the double-positive (DP) CD4+8+ phenotype, which is associated with low level expression of the α/β heterodimer TCR (TCRlow). Three pathways are possible outcomes for these cells: recognition of self-peptide/self-MHC (an autoreactive phenotype), which results in cell death by apoptosis; failure of recognition, which results in death by a default pathway; and positive selection, which results in expansion. The final one of these is accompanied by lineage separation into distinct CD4+8– and CD4–8+ populations, each of which expresses high levels of α/β TCR (TCRhi). The final maturation step is associated with migration to the thymic medulla, from which the mature cells emigrate to the periphery. Only about 2% of the cells that arise in the thymus emigrate to colonize the periphery; the remainder die in situ.

The three defining characteristics of the acquired response—specificity, memory, and self/nonself discrimination—reflect the properties and activities of lymphocytes. These cells are directly responsible for the acquired immune response, although in many instances the response also requires the involvement of cells of the monocyte/macrophage lineage (Unanue and Allen, 1987). For the investigator interested in applying immunological techniques to his or her work, the acquired immune response and the functions and interactions of lymphocytes, both with other lymphocytes and with other cell types, are of paramount importance. The remainder of this chapter will focus on these interactions.

III ACQUIRED IMMUNITY HAS TWO EFFERENT LIMBS

Two distinct types of response are evident in acquired immunity: humoral immunity, which is characterized by antibody molecules as effectors (Kuby, 1992), and cell-mediated immunity (CMI), which does not involve antibodies but is a local phenomenon, depending on close proximity between the stimulus, the effector cells, and the targets (Kuby, 1992). The two types of response reflect two separate lineages of lymphocytes: B cells for humoral immunity and T cells for CMI. Since antibodies are soluble glycoprotein molecules, they become distributed throughout the plasma and, in some instances, the extravascular space; thus, they can confer protection at a distance from their site of synthesis and secretion. Humoral immunity is effective in protection against extracellular bacteria and their products, such as toxins, and against re-infection by viruses. In contrast, the cells involved in CMI synthesize and secrete biologically active molecules, but these function only over short distances. In terms of CMI effector function, one can identify two separate activities: delayed type hypersensitivity (DTH) and cytotoxic T (Tc) cell activity. DTH and much of the Tc effector functions are properties of distinct subpopulations of T cells, which are distinguishable by their expression of particular cluster designation (CD) markers. DTH is a property of cells that express the surface glycoprotein CD4, whereas classical Tc cells express the CD8 glycoprotein (Reinherz et al., 1979). More recent work has indicated that, whereas only CD4 + cells mediate DTH, cytotoxic function is not restricted to CD8 + T cells but may also be demonstrated by CD4 + cells (Ju et al., 1990). In the periphery, that is, outside the thymus, the organ in which T cells develop, CD4 and CD8 expression are mutually exclusive, permitting unambiguous identification of the two subpopulations. DTH responses are protective against infection by intracellular pathogens, such as Mycobacteria and Listeria (Kaufmann, 1993). The response involves both specific T cells, referred to by some authors as Td cells because of their involvement in DTH, as well as additional recruited T cells and macrophages. The latter are attracted to the site of reaction and are activated in situ. The Tc response is manifest by the antigen-specific effector cells alone. These cells are able to bind to and kill their targets specifically and directly. Potential targets for Tc activity include virus-infected cells, cells in a tissue graft from a nonidentical donor, and autochthonous tumor cells (an autochthonous tumor is one that spontaneously develops in the animal, in contrast to one that is transplanted in as part of an experimental protocol).

Figure 2 indicates the separate effector pathways, a separation that originates at the level of their development in the primary lymphoid organs.

FIGURE 2 Two parallel effector arms, humoral and cell-mediated immunity, exist in the acquired immune response.

The generation of most humoral responses and both facets of CMI requires collaborative interactions between different cell types. Immune responses are generated by the activation of resting lymphocytes, followed by proliferation and differentiation to effector function (Kuby, 1992). Populations of memory cells are also generated during these events, the mechanism possibly involving, but not being limited to, failure to undergo terminal differentiation (Sprent, 1994). Central to proliferation and differentiation is the activity of T helper (Th) cells. These T lymphocytes are characterized by the expression of the CD4 surface marker (Reinherz et al., 1979). Depletion of CD4-bearing T cells leads to profound immune deficiency, as best exemplified by the acquired immunodeficiency syndrome (AIDS), in which infection by the human immunodeficiency virus type 1 (HIV-1) leads to a progressive loss of CD4-bearing lymphocytes (Fauci, 1993). To generate most humoral responses, antigen-specific B and Th cells are required (Parker, 1993). As noted earlier, T cell function operates only over short distances. In this instance, the need for close apposition of B and Th cells reflects the properties of the antigen-specific receptor on Th cells and the mechanisms by which help is delivered to B cells. Both these issues are dealt with in greater detail in subsequent discussion.

The immune system has evolved so most responses require the participation of more than one cell type. Although lymphocytes are responsible for memory, specificity, and self/nonself discrimination, they cannot function alone but require cells such as macrophages and dendritic cells, examples of so-called accessory cells. Accessory cells are essential to a number of aspects of the response, including the processing and presentation of foreign substances, so lymphocytes can recognize and respond to them; the delivery of activation signals to lymphocytes; and, in some instances, effector function proper, as a consequence of being activated by lymphocytes. Few immune responses, especially those that occur in response to replicating pathogens, take a single form, for example, being solely a humoral response. The complexity of the immune response, particularly as we know it to exist in mammals, reflects the interplay of multiple cell types. This interaction has the consequence of allowing several forms of immune response to occur simultaneously, thus maximizing protection for the organism.

A The Fate and Processing of Immunogen and Clonality Are the Keys to Understanding Acquired Immunity

The material used for immunization of an animal is called the immunogen. The capacity of a molecule to induce an immune response is referred to as its immunogenicity. Of the four classes of molecules that are used as immunogens, proteins and carbohydrates are generally strong immunogens, eliciting vigorous responses, whereas nucleic acids and lipids are both significantly weaker. Naturally occurring immunogens, such as replicating pathogens or vaccines derived therefrom, may incorporate more than one of these components, depending on their method of preparation. The word antigen is often used as a synonym for immunogen. Strictly speaking, the two words immunogen and antigen have different meanings, an antigen being a substance that can be bound by antibodies and an immunogen being a substance that is capable of eliciting an immune response. However, in general the two words are used interchangeably.

Several factors contribute to immunogenicity and to the efficiency of an immunization regime. The route of immunization determines the source of the responding cells. Immunization by the intramuscular, intravenous, or subcutaneous routes elicits a response in the systemic immune system, major response sites of which are the spleen and peripheral lymph nodes. The secretory immune system functions in parallel to the systemic, and comprises the lymphoid tissues associated with the gastrointestinal, bronchial, and urogenital tracts, as well as other glandular tissues such as mammary glands and tear glands. Secretory immunity is important for protection at mucosal surfaces (Underdown and Schiff, 1986). The principal route of immunization for the secretory immune system is by ingestion and, although it is infrequently employed by nonimmunologists for the production of reagents for experimental use, its importance in the homeostatic function of the immune system should not be ignored. Immunization of the systemic system by intramuscular, intravenous, or subcutaneous administration is routinely used for experimental purposes. Intramuscular and subcutaneous immunization give rise to responses initially in the lymph nodes draining the site of injection. Material introduced into the bloodstream is filtered by the spleen, which is the initial site of response. In each case, the immunogen follows a similar pathway, the steps of which are uptake, processing, and presentation (Brodsky and Guagliardi, 1991).

Uptake is the process by which the complex immunogen is ingested by those cells that are capable of inducing Th cell responses, including macrophages and B cells. For macrophage uptake, the uncomplexed immunogen may be ingested as such or, if there has been a prior immune response, it may be aided by IgG antibodies and by one of the split products of complement activation, C3b. This event is shown diagramatically in Figure 3. The ability of antibody and complement to potentiate immunogen uptake is called opsonization. Since B cells are lymphocytes, they express clonally distributed, antigen-specific receptors, their membrane immunoglobulin (mIg) molecules that are vehicles for the uptake of specific immunogen (Lanzavecchia, 1990). The immunogen-containing phagosome fuses with a lysosome and the immunogen–Ig complex is digested by the hydrolytic enzymes. Figure 3 also shows immunogen uptake by specific B cells, which utilize the endosomal pathway for uptake. From this point on, the immunogen follows similar pathways in both B cells and macrophages. The digestion of the protein components into individual peptides is referred to as antigen processing (Brodsky and Guagliardi, 1991). Particular emphasis is placed on the processing of proteins into peptides, because these are the principal (if not the only) moieties to which T cells can respond. Antigen presentation to Th cells requires the formation and membrane expression of complexes between processed peptides and glycoprotein molecules encoded by the class II region of the major histocompatibility complex (MHC) (Brodsky and Guagliardi, 1991; Germain, 1994). The complexes are formed intracellularly between newly processed peptides from the immunogen and newly synthesized class II MHC molecules (Germain, 1994). On formation, the complexes are transported to the membrane where they serve as ligands for the specific receptors on Th cells.

FIGURE 3 Antigen presentation to T cells requires a complex of processed peptide associated with an MHC glycoprotein. Presentation to CD4+ T cells requires peptide, derived from exogenous protein, presented in association with class II MHC gene products (upper illustrations). Both B cells and macrophages are capable of this, although they differ in their modes of protein uptake. B cells (upper left) employ specific binding by mIg to endocytose the protein–mIg complex. Macrophages (upper right) phagocytose proteins that are coated (opsonized) with either Ig alone or Ig plus a fragment of the complement protein C3, designated C3b. Presentation to CD8+ cells requires the peptide to be associated with class I MHC glycoproteins. The peptides presented by this pathway are derived by the cytoplasmic degradation of proteins that have been synthesized by the presenting cell (lower illustration).

In contrast, Tc cells have evolved to kill cells that have become altered in some way, for example, by viral infection. As for Th cells, a peptide–MHC complex is required for recognition, but a major difference exists because the peptides are synthesized by the presenting cell (Brodsky and Guagliardi, 1991; Germain, 1994). A consequence of the endogenous source of peptides is that their pathway to the membrane is different from that of peptides from an acid compartment. They form complexes and are expressed in association with glycoprotein products of the class I region of the MHC (Brodsky and Guagliardi, 1991; Germain, 1994); this event is also shown in Figure 3. Since class I MHC gene products are expressed on a far wider range of cell types than are class II molecules, many more cell types can activate, and be targets for, Tc cells. The class of MHC molecule with which peptide is expressed thus determines which T cell subpopulation will respond.

Lymphocytes are stimulated to respond by recognizing structures on the immunogen that are complementary to their receptor binding sites. The specificity of the effector phase of an immune response therefore reflects the activation of those lymphocytes that are stimulated. The model that describes this process is clonal selection (Burnet, 1959). This model postulates that, although lymphocytes originate from a limited number of progenitor cells, as they develop they diversify into a large number of independent clones. Clonal diversification occurs prior to the introduction or recognition of immunogen. Each clone of lymphocytes bears receptors of a single binding specificity on its surface. An immunogen introduced into an animal selects only those clones that bear receptors capable of binding structures on the immunogen molecule. Such structures are called determinants or epitopes. Although clonal selection occurs in both B and T cells, the binding characteristics of the receptors on the two cell types are markedly different. Table II compares the receptors on B and T cells, in terms of both structure and function. B cell receptors are immunoglobulin (Ig) molecules, the binding sites of which can recognize both conformational and sequential epitopes. Ig molecules are capable of binding free immunogen, or fragments thereof, in solution or on cell surfaces. In contrast, T cells express a heterodimeric receptor molecule, generically called T cell receptor (TCR). TCRs bind only sequential epitopes and are incapable of binding free immunogen. Binding by the TCR requires a proteolytically processed peptide fragment of a protein (or glycoprotein) immunogen, which must be presented to the TCR as a complex of peptide in association with MHC molecules (see preceding discussion). This requirement is referred to as MHC restriction of T cells (Carbone and Bevan, 1989). MHC molecules are, themselves, integral membrane glycoproteins and their expression is absolutely required for any cell type to function as an antigen-presenting cell (APC) to a T cell. As noted earlier, two populations of T cells are identifiable in the periphery on the basis of expression of mutually exclusive expression of CD4 and CD8. MHC class I and class II molecules differ in both structure and cellular expression, as shown in Table III. The binding requirements of the TCRs on the two subpopulations, CD4 + 8 – and CD4 – 8 +, differ with respect to the class of MHC molecule with which the processed peptide must be associated. The TCRs on CD4 + 8 – cells require the peptide to be associated with class II MHC molecules (Carbone and Bevan, 1989). Association with class I MHC is required for recognition by CD4 – 8 + cells (Carbone and Bevan, 1989). Class II molecules are heterodimers that are constitutively expressed on the membranes of only a relatively small number of cell types, including dendritic cells, B cells, and a fraction of the macrophage-lineage cells in a variety of tissues such as the Kupffer cells of the liver. Other cell types may be induced to express class II MHC, driven by stimuli such as interferon γ (IFN-γ) (King and Jones, 1983). Only those cells that express class II MHC can function as antigen-presenting cells for Th