CHAPTER 2 ANTIGEN/ANTIBODY INTERACTIONS

See APPENDIX (1) THE PRECIPITIN CURVE; (2) LABELING OF ANTIBODIES The defining characteristic of HUMORAL immune responses (which distinguishes them from CELL-MEDIATED responses), is their ability to be transferred by serum, and the proteins within serum which are responsible for such immunity are ANTIBODIES. We can formulate intriguingly circular definitions for antibodies and ANTIGENS, and note that the universal property of antibodies is their ability to specifically bind their cognate antigens. The consequences of such binding, however, can vary considerably, depending on the nature of the particular antigen and antibody involved. We distinguish the PHYSICAL and the BIOLOGICAL PROPERTIES of antibodies, and the properties of ANTIGENICITY versus IMMUNOGENICITY, and introduce the concept of ADJUVANTS, substances which are capable of increasing immunogenicity.

We'll begin by defining three important terms: ANTIBODY - The molecule present in serum and other body fluids which mediates humoral immunity, and which can bind specifically to an antigen. Serum which contains antibodies (directed against one or more antigens) is termed an antiserum. ANTIGEN - A molecule which can be specifically bound by an antibody (typically a protein or carbohydrate recognized as "foreign"). EPITOPE (= antigenic determinant = "antigenic specificity") - The minimum target structure on an antigen which is bound by a particular antibody molecule. A particular antigen molecule may (and generally does) bear many different epitopes or determinants, each of which can be a target for antibody binding. (NOTE: Antibodies themselves can serve as antigens; human antibodies, for instance, are "foreign" to rabbits, and can elicit rabbit antibodies to human antibody molecules. As we will see later, the use of antibodies as antigens has been an extremely powerful tool for understanding antibody structure and genetics.)

DEFINING HUMORAL IMMUNITY Experimentally defining a humoral immune response involves demonstrating that such immunity can be transferred by serum (or other fluids). The example below (Fig, 2-1) illustrates some key features of humoral immunity.
Live Pneumococcus Naive

DIES

Killed Pneumococcus Naive wait 2 weeks

Live Pneumococcus Immune

SURVIVES

"Active Immunity" Live Pneumococcus Naive "Passive Immunity"

Immune

TRANSFER SERUM

SURVIVES

Figure 2-1 If a mouse is injected with a sufficient dose of live Pneumococcus bacteria, it will die of infection within a few days. If, however, it has previously been injected with killed organisms, not only does it not succumb to infection, but it will survive a subsequent injection of a normally lethal dose of this organism; such a mouse has been immunized, and is therefore said to be immune to Pneumococcus. Although not illustrated here, we can further demonstrate that this resistance is specific the immune mouse will retain normal susceptibility to some other organism to which it had not previously been exposed. Such specificity establishes that the immunity we see is a result of the mouses adaptive immune response. Question: Does this resistance represent humoral immunity? To find out, we take serum from the immune mouse and inject it into a non-immune recipient, then inject a lethal dose of Pneumococcus. We find that this recipient survives this treatment; serum from an immune mouse transfers immunity to a nave recipient. This demonstrates that immunity to this organism is mediated by humoral immunity. (NOTE: This does not, however, mean that resistance to all bacterial infections is mediated by humoral immunity. As we will see in Chapter 12, transferring serum from a mouse which is immune to another bacterium, Listeria (which is an intracellular pathogen), does not confer resistance to nave recipients; such immunity is therefore not humoral.) This illustration also serves to define two distinct modes of adaptive immunity, namely ACTIVE IMMUNITY and PASSIVE IMMUNITY. Immunization of the mouse in the second line of Fig. 2-1 results in a state of "active" immunity; the animal's own immune system is responsible for resistance to the subsequent bacterial challenge. On the other hand, transfer of serum, as in line 3 above, results in a state of "passive" immunity in the recipient; such immunity is the result of the presence of transferred antibody (see below). The animal's own immune system does not participate at all, and this immunity lasts only as long as sufficient levels of antibody are present. 10

The substance present in immune serum which is responsible for transferring immunity is antibody. In addition to transferring resistance to infection, these serum antibodies can carry out a variety of other functions. For example, if immune serum is mixed with a suspension of Pneumococcus, the bacteria will be seen to rapidly "clump" together. This effect is known as agglutination, and is one of the many ways in which antibodies can be detected and quantitated. The various effects that antibodies may exhibit can be generally categorized as physical effects, which depend only on the physical nature of the antibody and antigen, or biological effects, which additionally depend on the particular biological properties of the target antigen or other biologically active molecules which are involved. PHYSICAL EFFECTS OF ANTIBODY Agglutination. "Clumping" of a particulate antigen, e.g. bacteria or SRBC (sheep red blood cells). Agglutination of red blood cells is a technique which has been widely used in clinical and basic research as well as in the clinical laboratory, and is called HEMAGGLUTINATION. Many soluble antigens can be made effectively particulate by coating them onto SRBC or latex or other particles; the resulting clumping by antibody is known as passive agglutination. Precipitation. Interaction of antibody with a soluble antigen to form an insoluble complex, e.g., with BSA (bovine serum albumin). In liquid - the precipitate can be recovered by centrifugation and analyzed (see APPENDIX 1, THE PRECIPITIN CURVE). If either the antigen or antibody is radioactively labeled (see APPENDIX 2, LABELLING OF ANTIBODIES), it can be used in a RadioImmunoPrecipitation (RIP) assay, first developed in the 1950s. In agarose - if the antigen-antibody interaction takes place in a semi-solid medium such as agarose, the resulting precipitate can be easily visualized. This is of special significance in a configuration known as Ouchterlony Analysis (see APPENDIX 3, OUCHTERLONY ANALYSIS). Precipitation and agglutination are both consequence of cross-linking of antigens by antibody into large complexes. The ability of antibodies to carry out this process implies that each antibody can bind at least two antigen molecules, and that it can only occur if the antigen molecule has two or more epitopes (determinants ") which can be recognized by that antibody. Binding. If an antigen is bound to a solid matrix (latex particles or a plastic dish, for example), and if the antibody is labeled in some way (with a visible, radioactive or enzyme molecule), binding of the antibody to its antigen can be easily and sensitively measured. If a radioactive label is used, the assay is called a solid-state RadioImmunoAssay (RIA). With an enzyme-based label, on the other hand, it becomes an Enzyme-Linked ImmunoSorbent Assay (ELISA). These solid state assays (particularly ELISA's) have largely replaced precipitation and agglutination assays in a wide variety of clinical and research applications.

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BIOLOGICAL EFFECTS OF ANTIBODY Protection from infectious disease. We have already seen in the Pneumococcus example (Figure 2-1) how this manifestation of antibody can be assayed by transferring serum from one animal to another. Immobilization. An antibody directed against components of the flagellae of motile bacteria or protozoa can cause these flagellae to stop moving. This results in the loss of the organisms' ability to move around, and this loss of motility can be detected by microscopic examination. Cytolysis. If the target antigen is an integral component of the membrane of certain sensitive cells, antibodies may cause disruption of the membrane and death of the cell. This requires the participation of a collection of other serum components collectively known as COMPLEMENT (see Chapter 5), and binding of these components to antibodies is referred to as Complement Fixation. If the antigen target is a red blood cell, this effect is known as hemolysis, which can be readily detected visually. In the case of a bacterial cell target, the effect is referred to as bacteriolysis. If the target is a nucleated cell the effect is referred to as cytotoxicity, and may be measured by release of a radioactive label incorporated into the cell (such as 51Cr), exclusion of "vital" dyes such as Trypan Blue, or any of several other measures of cell viability. Opsonization. If the target antigen is particulate (e.g. a bacterium, or an antigencoated latex particle), bound antibodies may greatly increase the efficiency with which the particles are phagocytosed by macrophages and other "scavenger" cells. This improvement of phagocytosis is known as opsonization, and may be facilitated even further by the presence of complement. As will be discussed later, opsonization is the result of antibodies increasing the degree to which antigenic particles will "stick" to phagocytic cells. This phenomenon has therefore been referred to as immune adherence, and depends on the presence in the membranes of white blood cells of specific receptors either for antibody (FcR, or "Fc-receptors") or for complement (CR, or "complement receptors"), both of which will be discussed later (see Chapter 14, for example). ONE COMMON DEFINING PROPERTY OF ANTIBODIES: ALL ANTIBODIES EXHIBIT SPECIFIC BINDING TO ANTIGEN Different antibodies may show various combinations of effects; some antibodies may precipitate but not interact with complement (and therefore not show cytolysis), some may be opsonizing but not be capable of agglutination. The single common feature of all antibodies, however, is that of specific recognition and binding to antigen. All other effects, physical or biological, are secondary consequences of this specific binding. The structure of antibodies and the basis of their ability to specifically bind antigen are the subjects of the next two chapters (Chapters 3 and 4).

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ANTIGENS, IMMUNOGENS AND HAPTENS We have been discussing "antigens" as molecules (1) which can elicit antibody production upon injection into an appropriate host; and (2) to which these antibodies can then bind. The difference between these two properties is an important one which we will now make explicit by defining two related but distinct terms: IMMUNOGEN. A molecule which can elicit the production of specific antibody upon injection into a suitable host. ANTIGEN. A molecule which can be specifically recognized and bound by an antibody. These definitions imply that an immunogen must be an antigen, but an antigen is not necessarily an immunogen. Lets illustrate this in the following table: Substance 1) BSA 2) DNP 3) DNP10-BSA 4) "clarified" BSA Molecular weight "68,000" ~200 "70,000" 68,000 Immunogen? yes no DNP - yes BSA - yes no (see Note) Antigen? yes yes yes yes yes

If we take a conventional preparation of purified bovine serum albumin (BSA) and inject it into a mouse (line 1 in the table above), the mouse will produce antibodies which will bind to BSA. BSA is therefore both an immunogen and an antigen. If we take the small organic molecule dinitrophenol (DNP) and inject it into a mouse (line 2), no antibodies will be produced which can bind DNP. DNP is therefore not immunogenic; we will deal with its antigenicity shortly. We can chemically couple DNP molecules to the protein BSA, yielding DNP-BSA. If we inject this material into a mouse (line 3), we see that antibodies to BSA are elicited (as we would expect), but also find antibodies which will bind specifically to the DNP groups on BSA; we can further demonstrate that these anti-DNP antibodies will also bind free DNP (or DNP coupled to any other molecule). Therefore, DNP-BSA is both immunogenic and antigenic (with respect to both the DNP groups and the BSA itself), and the free DNP is also antigenic, even though we have shown it is not immunogenic. DNP is an example of a HAPTEN, a small molecule which is not immunogenic unless it is coupled to a larger immunogenic CARRIER molecule, in this case BSA. (Such a hapten/carrier system will be used in Chapter 14 to illustrate the mechanisms of cell interactions required to generate humoral immune responses). We can further demonstrate that the immunogenicity of BSA depends on the presence of aggregates of BSA molecules. If we take a sample of our BSA and centrifuge it at high speed we can remove any aggregated material, leaving behind only single, monomeric BSA molecules in solution. If we immediately inject this "clarified" BSA into a mouse we find that it does not elicit the production of antibodies (as seen in line 4); this monomeric BSA is 13

therefore not immunogenic (nor can it serve as an effective carrier for a hapten). It is still antigenic, however, which we can show by reacting it with the anti-BSA antibodies which we made against the non-clarified BSA (in line 1, for example). (NOTE: "Clarified" BSA not only fails to induce antibody formation, but can induce a state of TOLERANCE to BSA, defined as the specific inability of the mouse to respond to subsequent injections of normally immunogenic BSA. The mechanism of such tolerance will be discussed in Chapter 18.) REASONS FOR LACK OF IMMUNOGENICITY Substances may lack immunogenicity for a variety of reasons: 1) Molecular weight too low. Haptens, for example, are not immunogenic until they are coupled to a high molecular weight carrier. There is no simple cutoff for required molecular weight, however; we have already seen that even the 68,000 mw of BSA is not sufficient to be immunogenic unless the molecules are aggregated into even larger complexes. 2) Not foreign to host. The adaptive immune system normally responds only to "foreign" substances. A sheep, for instance, will normally not make antibodies against its own red blood cells (SRBC), although SRBC are highly immunogenic in mice. (The basis of normal SELF-TOLERANCE is covered in Chapter 18). 3) Some molecules are intrinsically poor immunogens for reasons which are not well understood. Lipids, in general, are poor immunogens, probably because they do not have a structure rigid enough to be stably bound by antibodies. Nucleic acids are also relatively weak immunogens, although they are nevertheless common targets for antibodies present in various autoimmune diseases (discussed in Chapter 19) HOW TO INCREASE IMMUNOGENICITY: ADJUVANTS (See also CHAPTER 22) An ADJUVANT is any substance which, when administered together with an antigen, increases the immune response to that antigen. One of the most widely used adjuvants (in animals but not in humans) is FREUND'S ADJUVANT, which consists of mineral oil, an emulsifying agent, and killed Mycobacterium (the organism which causes tuberculosis). A solution of the desired antigen in water or saline is homogenized with this oil mixture, resulting in a water-in-oil emulsion which is injected into the recipient. Its powerful adjuvant properties result from several factors: 1) The antigen is released from the emulsion over an extended period of time, causing a continuous and more effective stimulation of the immune system. (Antigen given in soluble form will typically be cleared in a matter of hours or days, whereas it can persist for weeks or months in a depot created by the adjuvant.)

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2) The Mycobacteria contain substances which non-specifically stimulate the immune system, resulting in a higher level of response to the specific antigen. One of these substances which has been extensively studied is Muramyl Dipeptide (MDP). Although Freund's Adjuvant is not used in humans, other forms of adjuvant can be used, such as alum precipitation of antigen, by which a soluble antigen is precipitated together with aluminum hydroxide, resulting in particles of the salt coated with antigen. A soluble antigen is thus converted to a particulate form, and again is released from the mixture over an extended period of time. Substances such as purified MDP and others are also being used to develop effective adjuvants which are less toxic, and therefore potentially usable in humans (see Chapter 22)

CHAPTER 2, STUDY QUESTIONS: 1. 2. 3. 4. 5. Define ANTIBODY, ANTIGEN, IMMUNOGEN and HAPTEN. How would you determine if a particular immune response is a HUMORAL response? Describe assays which could be used to measure AGGLUTINATION, PRECIPITATION, HEMOLYSIS and OPSONIZATION. Describe two antibody assays which require no antibody function other than specific binding to an antigen. Define and distinguish ACTIVE versus PASSIVE immunity.

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