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  • 1. MBBS 1st Yr. Lecture Dr. D. Higgins October 22, 2002 - 8:30 AM Underground Lecture Theatre 1, New Clinical Building INTRODUCTION TO HEALTH AND DISEASE BLOCK IMMUNOLOGIC SPECIFICITY The immune system recognizes foreign antigens with an amazing degree of accuracy – i.e. specificity. The purpose of this lecture is to discuss what it is that the immune system recognizes, how we can define immunologic specificity, and what is the basis of specificity in terms of structure of the immune recognition modules. The question of immunological specificity has been discussed since long before the structure of antigens and antibodies was known. The father of the historical concepts was Paul Ehrlich. In the 1870s and 1880s he developed theories of immunological interaction most of which hold today. Ehrlich believed that the specificity of the immune system was based on stereochemical fit between antibodies and antigens, just as a toxin fits to a receptor; the geometry of this stereochemical interaction was precise - rather like a “lock and key” - such that only one antigen- antibody combination could match up. It was a further 80 years before Ehrlich’s predictions could be confirmed by 20th century protein analysis. ANTIGENS AND THEIR EPITOPES First, it is essential to understand some basic principles of antigen structure. Almost all strong antigens are large (> 10,000 daltons) proteins. The strongest are the glycoproteins associated with particles such as bacteria, viruses, genetically different cells; soluble proteins are less strongly antigenic than those associated with cell structures. Within a protein antigen, there are numerous sites that will each be recognized. These sites are called HAPTENS or EPITOPES. Each epitope is 6-9 amino acids or 6-9 carbohydrate residues. Different epitopes in one protein During the immune response, each epitope will be responded to by a unique set of T cells and B cells. Thus, the intact protein is not a single epitope but a collection of different epitopes. A large glycoprotein, ± 100,000 daltons, will possess hundreds of epitopes and will stimulate hundreds of B and T cell clones. 1
  • 2. From this, it can be seen that there are two levels in which antigens can display similarity. 1. Two different cells might carry the same surface antigens (A). They will also carry unique antigens (B, C): If we were to raise antisera to these cells in, say, rabbits the antisera would contain common antibodies (to A) and unique antibodies (to B or C). Totally specific sera would recognize either B or C, but not A. 2. Two different proteins might contain the same epitopes: An antiserum that recognizes all the epitopes on a protein is "polyclonal". An antiserum that recognizes only the unique epitopes is either "monospecific" or "monoclonal" depending how it is made. 2
  • 3. Epitope distinction Immunologists realized the concept of epitopes within large proteins many years ago and used this principle to their advantage. They found that if they linked a small side group (e.g. a benzene ring-based compound) to a protein, they could then use the unconjugated side group to study antibody formation. Thus it was found that haptens will stimulate antibody formation if they are conjugated to a carrier protein, that the unconjugated ha pten will bind to anti-hapten antibodies but that the unconjugated hapten would not stimulate antibody formation. Examples of haptens used in the study of antibody formation and specificity include: 1. Arsanilic acid This can be diazotized and coupled to proteins via the tyrosine amino acids. 3
  • 4. 2. Dinitrofluorobenzene This couples spontaneously (under conditions of high pH) to the lysine residues of protein. The new side-arm is called the “dinitrophenyl hapten”, or DNP for short. These synthetic haptens have been used to determine many properties of the antigen-antibody interaction, including the size of the antigen binding cleft on the antibody, and numerous aspects of the concept of specificity. The typical use of the DNP hapten is illustrated below. A protein (A) is conjugated with DNP. The result is that it retains all its usual epitopes and gains DNP. When the protein is injected into an experimental animal, the resulting antiserum recognizes all the protein epitopes and the DNP. To study the anti-DNP antibodies in isolation, we need a second, unrelated protein (B), which we also conjugate with DNP. Now, the only interaction between the antiserum and the protein is between DNP epitopes and anti-DNP antibodies, hence, with appropriate assays and some smart mathematics, the kinetics of a single interaction can be determined. 4
  • 5. 1. 2. 5
  • 6. In one of the classic experiments concerning specificity, Landsteiner immunized rabbits with meta-aminobenzene sulphonic acid. H tested the resulting serum and made the following e observations: 1. The antibodies did not react with the structurally similar compounds meta-aminobenzene arsonate and meta-aminobenzene carboxylate. 2. When tested against the ortho-, meta-, and para- configurations of benzene sulphonic acid, the serum raised against meta- reacted strongly with meta- and weakly with ortho- and para-. Similarly, if rabbits were immunized with the ortho- or para- derivatives, the resulting antiserum invariably reacted most strongly with the configuration used for inoculation. The conclusion here is that antibody can differentiate very finely between haptens that are chemically and structurally similar. THE ANTIGEN-BINDING SITE Three types of structures bind haptens specifically: antibody (immunoglobulin), TCR, and MHC. In each case, the hapten-binding cleft is formed between the convolutions of two domains. There are many similarities, but some differences, between these three systems. Here, we will concentrate on the Immunoglobulin/antibody system. 6
  • 7. The antigen-binding sites of an immunoglobulin are each made up of a VH and VL domain. Variability is determined by the amino acid sequence of the domains – every specificity of antibody will have a different seque nce for the ± 110 amino acids within the VH and VL domains. The general level of variability through the VH and VL is about 15% but there are certain areas – 3 in the VL, 4 in the VH – where much greater variability occurs. These are called HYPERVARIABLE or COMPLEMENTARITY – DETERMINING regions (CDR); the areas of lower variability are called FRAMEWORK regions (FR). The 3 -dimensional convolutions of the VH and VL are such that the CDRs project outwards, rather like fingers, and so make a cup into which the hapten will fit. The specificity of this cleft for hapten is determined by two factors: 1. Amino acid sequence. The amino acid sequence of the CDRs of the H and L chains must be complementary to the structure of the hapten. 2. Shape. The configuration of the antigen binding site must match that of the hapten – "lock and key" concept. Binding between antibody (also TCR and MHC) and hapten is non covalent. It involves several forces: Hydrogen bonding Electrostatic forces Van de Waals for ces Hydrophobic forces 7
  • 8. The applications of these forces require close contact between hapten and antibody, i.e. a good fit. If the fit is good, there will be strong binding – high AFFINITY If the fit is poor, there will be weak binding: In general, the best and most specific antibodies have a high affinity of binding. 8