CHAPTER 3 ANTIBODY STRUCTURE I
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1 CHAPTER 3 ANTIBODY STRUCTURE I See APPENDIX: (3) OUCHTERLONY ANALYSIS; (6), EQUILIBRIUM DIALYSIS; (7) CROSS-REACTIVITY Electrophoretic separation of serum proteins identifies the GAMMA-GLOBULIN fraction as containing the majority of antibodies. Three terms which are often confusingly interchanged are defined and distinguished (GAMMA-GLOBULIN, IMMUNOGLOBULIN, ANTIBODY), as are two terms describing antibody/antigen binding, AFFINITY and AVIDITY. All antibodies are made up of one or more IgG-like subunits, each of which has exactly two antigen-combining sites. The affinity of these sites for their antigen (defined as the K eq of the binding reaction) is highly heterogeneous in any normal immune response. While the avidity of an antibody (its ability to form stable complexes with antigen) does depend on its intrinsic affinity, it also increases dramatically with an increasing number of combining sites per antibody. In order to determine the structure of antibodies, we first must have a way of isolating these molecules in relatively pure form. We ll begin by describing the general process of serum fractionation, then go on to analyze the nature of antigen-antibody binding. The many components of normal serum can be separated from one another by various means: Salt precipitation. Ammonium sulfate [(NH 4 ) 2 SO 4 ] as well as a variety of other salts can be used to precipitate serum components; different proteins will precipitate at different concentrations of salt, providing a convenient means of separating them. The fraction containing most of the antibody activity generally precipitates at relatively low salt, at about 30-40% of saturated ammonium sulfate. This is a very widely used experimental method for fractionation of serum components (and proteins in general). Ethanol precipitation. Ethanol can also be used to precipitate serum components, which come out of solution at different concentrations and under different conditions of ionic strength, ph and temperature. This is a more elaborate procedure to carry out than salt fractionation, but is the basis for Cohn Fractionation, which in modified form remains a standard procedure for preparing serum protein fractions for clinical use more than sixty years after its original description in the 1940 s. Electrophoresis. Different serum proteins migrate at varying rates in an electric field, a property which can be used to separate them. While this procedure can be adapted for use on a preparative scale, it is most commonly used for analysis. 17
2 A typical pattern generated by electrophoresis of a serum sample (e.g. on a filter paper strip) is shown in Figure 3-1. albumin globulins α1α2 β γ ELECTROPHORESIS OF NORMAL SERUM Figure 3-1 Several important points emerge from this pattern: 1) Most serum proteins carry a negative charge, and therefore tend to migrate from the point of origin (labeled "O") toward the anode, the positively charged electrode. 2) Four major peaks are seen in this example; these are named (from the anodal, or positive, side) the albumin peak (which is by far the largest), followed by four globulin peaks, α1- and α2-globulin, β-globulin and γ-globulin. 3) This pattern is deceptively simple; serum actually contains hundreds of known proteins. Thus, "β-globulin" is not a single protein, but a mixture of many components which all happen to migrate in a particular region on electrophoresis. 4) Most (but not all) antibodies migrate in the γ -globulin region. 5) The γ-globulin peak is markedly broader than the others, reflecting the high degree of heterogeneity of the antibodies it contains. This heterogeneity is so great that some antibody molecules in fact migrate in the positions characteristic of α-globulin or β- globulin. 6) The γ-globulin peak is generally centered near the origin, labeled "O"; this reflects the fact that antibodies as a group are relatively neutral, i.e. less highly charged than most other serum components. 18
3 Three easily confused terms are all commonly used to refer to antibody molecules, gamma-globulins, immunoglobulins and antibodies. To avoid this confusion let's explicitly define each of them: GAMMA-GLOBULIN -- Any molecule which migrates in the gamma-globulin peak on electrophoresis. Most, but not all, antibodies are in this category, although the term is often used to refer to antibodies in general. (Other serum components migrate in this region as well; therefore, strictly speaking, not all gamma-globulins are antibodies.) IMMUNOGLOBULIN -- A family of molecules (to which all antibodies belong) with similar structures and physical properties. We shall see that these involve homologous amino acid sequences, similar "domain" structures and similar quarternary structures (the ways in which different polypeptide chains are joined into a larger functional unit). ANTIBODY -- A molecule belonging to the Immunoglobulin family, with binding specificity for a particular antigen. While all antibodies are immunoglobulins, most but not all antibodies are gamma-globulins. Note that our definition of "antibody" requires knowledge of the binding specificity of the molecule. If one is dealing with an "antibody" molecule whose specificity is not known, or is irrelevant, it is more accurate to refer to it simply as an "immunoglobulin". (Common usage of these terms varies considerably, however.) ANALYSIS OF THE ANTIBODY COMBINING SITE: VALENCY, AFFINITY AND AVIDITY If we immunize a rabbit with DNP-BSA, we can obtain an antiserum which contains antibodies to both the hapten and the carrier protein. This antiserum will precipitate DNP- BSA in addition to DNP-KLH (Keyhole Limpet Hemocyanin, an unrelated protein carrier). If we attach DNP to SRBC (sheep red blood cells) or to latex particles, we can show that the antiserum is capable of showing agglutination (and possibly hemolysis in the case of SRBC). We can use these antibodies to the DNP hapten in order to learn about antibody structure and function. Specifically, we will ask two questions: 1) How many hapten molecules can a single antibody molecule bind (i.e how many combining sites does it have, or what is its valency )? 2) What is the strength of binding of the hapten to its combining site(s) on the antibody molecule (i.e. what is the affinity of the combining site)? We have previously made the prediction that in order for an antibody molecule to be capable of precipitation or agglutination it must have at least two combining sites, in order to permit cross-linking of the antigen into large, insoluble complexes. We can determine the actual number of combining sites of our anti-dnp antibodies, as well as their affinity, by several techniques; one of them, EQUILIBRIUM DIALYSIS, is discussed more fully in APPENDIX 6, and we will use the results of such an analysis as the basis for our discussion below. 19
4 RABBIT IgG ANTIBODIES HAVE TWO HAPTEN-COMBINING SITES The structure of rabbit IgG antibodies represents the basic structure of all antibodies and we can show by equilibrium dialysis that each anti-dnp antibody molecule can bind exactly two DNP molecules. Thus, our minimum prediction of at least two combining sites is fulfilled. Other kinds of antibodies can be shown to have more than two combining sites (IgM and some IgA), but we will see that such antibodies are always made up of multiple units of the basic "IgG-like" structure, each of which bears precisely two combining sites. CONVENTIONAL ANTIBODIES ARE HETEROGENEOUS WITH RESPECT TO AFFINITY The DNP hapten is bound to each combining site by non-covalent forces, and the strength of this binding is measured by the equilibrium constant of the binding reaction, known as the AFFINITY. The antiserum we describe above contains anti-dnp antibodies with many different affinities, typically ranging from 10 5 to (Antibodies certainly exist with affinities outside this range, but such values are difficult to determine accurately due to technical limitations.) This antibody heterogeneity is a hallmark of the immune response, and has many practical and theoretical implications (see discussions of Clonal Selection and Affinity Maturation [Chapter 7], and Isotype Switching [Chapter 9]). The broadness of the gammaglobulin peak on serum electrophoresis (which we have already described) is one consequence of this heterogeneity; in fact, a sharp, narrow gamma-globulin peak (representing a homogeneous protein) is a pathological sign of a myeloma or other monoclonal gammopathy. However, homogeneous antibodies known as HYBRIDOMAS, or MONOCLONAL ANTIBODIES can be generated experimentally, and are important in many research and clinical applications (see APPENDIX 13). ANTIBODY AVIDITY: ABILITY TO FORM STABLE COMPLEXES WITH ANTIGEN AFFINITY is a thermodynamically defined term representing the strength of interaction of a single combining site with its hapten. Naturally produced antibodies always have two or more sites, however, so that affinity does not tell the whole story with respect to antigen-binding. A bivalent anti-dnp antibody, for example, can simultaneously bind to two DNP haptens on a single BSA molecule, resulting in a much more stable complex than if it only bound to a single site. AVIDITY, on the other hand, is the term used to describe the ability of an antibody to form stable complexes with its antigen. Avidity, of course, depends partly on affinity; all other things being equal (which they rarely are), one IgG antibody with a higher affinity for DNP than another will also have a higher avidity. However, various other factors also play a role, such as the number and spacing of the epitopes on the antigen, the distance between the combining sites on the antibody, and properties such as the "flexibility" of the particular antibody molecule. Avidity does not have a formal thermodynamic definition, and is most commonly used only in a relative context (by demonstrating that one antiserum may exhibit a higher or 20
5 lower avidity than another). Nevertheless, in discussing the interaction of an intact antibody (which is at least bivalent) with a conventional antigen (which is almost always highly multivalent), one must almost always think in terms of "avidity" rather than "affinity". This is of particular importance when considering the biological effectiveness of antibodies which have more than two combining sites, such as serum IgM and some IgA. CHAPTER 3, STUDY QUESTIONS: 1. Define the terms ANTIBODY, IMMUNOGLOBULIN and GAMMA-GLOBULIN. 2. How is EQUILIBRIUM DIALYSIS carried out, and what can it measure? 3. Define and distinguish antibody AFFINITY and AVIDITY. 21
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