Antigen structure and immunogenicity
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This document discusses the structure and function of antibodies and antigens. It defines key terms like antigen, immunogen, and antibody classes. The main classes of antibodies are IgG, IgA, IgM, IgD, and IgE. Each has a distinct structure and function. For example, IgG is the most common antibody in blood and can enter tissues, while IgA concentrates in body fluids to guard entrances. The document also explains how antibody structure relates to functions like complement activation, opsonization, and placental transfer of maternal antibodies to the fetus.
Antigen structure and immunogenicity
- 1. Antigen Structure and Immunogenicity An antigen is any substance that react with T or B lymphocytes. OR Substances which can be recognized by Ig of B cells (at Fab sites) and TCR’s of T cells (when accompanied by MHC) B and T cells also differ in the way they recognize Ag Recognition by B-cell and T-cell Receptors Antigen (Ag): Binds specifically to an antibody binding site (Ab), or to a T-cell receptor (TCR)* (* When the antigen is being presented to the TCR on a specific set of cell-membrane proteins called MHC). Immunogen: Binds specifically to an antibody binding site or to a T-cell receptor*, and generates a humoral or cellular immune response. All immunogens must be antigens, Not all antigens can generate a response. THERE ARE FIVE CLASSES OF ANTIBODIES BASED ON THE STRUCTURE OF THEIR HEAVY-CHAIN C DOMAINS All Antibody Classes Have Either l or k Light Chains As mentioned earlier, the two determinants used to define antibody light chains are called kappa (k) and lambda (l). Each chain has a molecular weight of about 23 kD and exists as one polypeptide chain of about 214 amino acid residues. The first 108 amino acids make up the V region, followed by roughly 110
- 2. A N T I B O DY S T RU C T U R E A N D F U N C T I O N amino acids that make up the C region. These two regions compose the VL and CL chain domains. The ratios of k to l light chains varies greatly within mammalian species. In mice, it is 95:5; in humans, it is about 60:40. However, there is no difference in either chain’s ability to pair with a heavy chain. IgG Is the Major Antibody in the Blood, but It Is Able to Enter Tissue Spaces and Coat Antigens, Speeding Antigen Uptake IgG, primarily induced by protein antigens, constitutes about 80% (12.5 mg/ml) of the antibody in serum. The or l) and two heavy chains (g). The four polypeptide IgG (150 kD) is composed of two light chains (either k chains are covalently held together by disulfide bonds. Human IgG consists of four subclasses (isotypes), which are numbered in order of their serum concentrations (IgG1, IgG2, IgG3, and IgG4). The four subclasses have 90 to 95% identity with each other in the C-region domains. The g chain is made up of four domains, one in the V portion and three in the C portion of the chain. The g1 chain is the shortest heavy chain, with 446 amino acid residues. On the CH2 domain (at position 297) of all g chains is attached one carbohydrate group that controls the quaternary structure of this domain. The chief distinguishing characteristic among the four IgG subclasses is the pattern of interchain linkages in the hinge region. IgA Concentrates in Body Fluids to Guard the Entrances of the Body Human IgA constitutes only 13% (2.1 mg/ml) of the antibody in human serum, but it is the predominant class of antibody in extravascular secretions. The IgA present in secretions (tears, saliva, nasal secretions, bronchial and digestive tract mucus, and mammary gland secretions) is secretory IgA. While the precise organization of secretory IgA is unknown, the model of current choice is depicted in Figure 4- 8. The J chain is a 15-kD polypeptide consisting of 129 amino acid residues and one carbohydrate group. It is synthesized by plasma cells and attaches to IgA (or IgM) either before or at the time of secretion. The J chain attaches to the carboxyl-terminal penultimate cysteine of either the a or the m chain. Dimeric IgA binds to the blood side of the epithelial cells through Fc receptors (Figure 4-9). These receptors are also called secretory, or S, proteins. Bound IgA is internalized and moves through the cytoplasm of the epithelial cells. IgA is detached from the cell following cleavage of S protein. The remaining peptide, called secretory component or piece, attaches to dimeric IgA. Depending on the species, it may or may not be disulfide-linked to the IgA dimer. It also gives resistance to enzymatic cleavage while in mucosal secretions. The a chain is made up of one V domain and three C domains. IgA1 is the most prevalent form in serum, but IgA2 is slightly more prevalent in secretions. Only IgA2 has allotypic determinants, and only the A2m(1) uniquely lacks interchain disulfide bridges between light and heavy chains. Instead, chains are linked to their own counterparts (one light chain to the other light chain). Another difference between IgA allotypes is the size of their hinge regions.
- 3. IgM Is the Largest Antibody; It Tends to Remain in the Blood, Where It Can Lead to Efficient Killing of Bacteria IgM, primarily induced by polysaccharide antigens, is a 950-kD pentamer that makes up about 8% mg/ml) of the antibody in the serum. The five monomeric IgM molecules are arranged radially, the Fab fragments pointing outward and the Fc fragments pointing to the center of the circle (Figure 4-10). IgM is the first antibody to appear during an immune response and the first formed by a developing fetus. Because of its many antigen-binding sites, IgM can quickly clump antigen and efficiently activate complement. IgM acts as one of the main receptors on the surface of mature B cells, along with IgD. When IgM is a surface receptor, it is in its monomeric form. The IgM m chain consists of 576 amino acid residues, with 452 making up the C region. Unlike g and a chains, which have three C-region domains, the m chain has four. The five carbohydrate groups are in the CH1 and CH3 domains and in the part of the m chain where the J chain binds. The CH2 domain of the m chain is equivalent to the hinge regions of the g and a chains. The m chain has two interchain disulfide bonds. The membrane form of IgM has a different carboxyl- terminal end. The membrane form of IgM is made up of 41 additional amino acid residues, of which 25 form a transmembrane segment of hydrophobic (nonpolar) amino acids followed by hydrophilic (polar) amino acids. IgD Remains Membrane-Bound and Somehow Regulates the Cell’s Activation IgD (175 kD) constitutes less than 1% (40 mg/ml) of the antibody in human serum. IgD is an antibody whose function remains unknown, even though it is one of the main receptors on mature B cells. As B cells mature, IgD is replaced by other antibodies. IgD may be a regulator of immune responses through its role in antigen internalization. The d-chain C region is divided into three domains and consists of 383 amino acid residues. The hinge region of IgD consists of 64 amino acid residues, longer than any other antibody class. IgE Is Found in Trace Amounts in the Blood, but It Still Triggers Allergies Human IgE (190 kD) makes up less than 0.003% (0.4 mg/ml) of the antibody in serum. IgE binds through its Fc part to mast cells or basophils. On later exposure to the same antigen, mast cells and basophils bind antigen with membrane-bound IgE and trigger allergic reactions. IgE protects against parasites by releasing mediators that attract eosinophils. Like the m chain, the e chain contains four C-region domains. IgE is made up of about 13% carbohydrate. The e chains are similar in size to m chains, except that e chains lack the 18 amino acid residues for J-chain binding. For further discussion of IgE,
- 4. THE BIOLOGICAL EFFECTOR FUNCTIONS OF ANTIBODIES ARE MEDIATED BY THE C DOMAINS Whereas a small part of the V region on an antibody determines the antigen specificity, single domains in the C region of heavy chains determine the effector functions. Biologic activities of antibodies divide into three general areas: (1) protection, (2) placental transfer, and (3) cytophilic (literally, “cell-loving”) properties. Although antibody binding blocks the attachment of toxins or viruses (called neutralization), antibodies alone cannot directly destroy a foreign organism. Instead, antibodies mark them for destruction by other defense systems. When IgM or IgG (except IgG4) binds to antigen, the complement system is activated and promotes bacterial lysis or accelerated phagocytic uptake. IgM or IgG molecules that have not reacted with antigen do not activate complement. IgM also mediates agglutination reactions. The coating (opsonization) of organisms with primarily IgG antibodies leads to enhanced phagocytosis by macrophages and neutrophils. Antibodies allow for the interaction of several cell types with antigen–antibody complexes through the cells’ Fc receptors. Because of their characteristic immunoglobulin-like extracellular domains, Fc receptors belong to the immunoglobulin gene superfamily (see Figure 9-4 in Chapter 9). Multiple biologic functions can be triggered through the crosslinking of any of the three Fc receptor classes. Macrophages have enhanced engulfment of antigen–antibody complexes through Fc receptors. B-cell Fc receptor engagement by antigen– antibody complexes regulates B-cell activation. Other cell types expressing Fc receptors (CD16) can use antibody- dependent cell-mediated cytotoxicity (ADCC), to lyse target cells coated with IgG. Certain antibodies, like IgA, can be localized to the lumens of mucosalined organs to provide mucosal immunity.
- 5. The second area of biologic activity associated with the antibodies is the movement of maternal antibody across the placenta to the fetus. The human fetus and newborns have limited immune responses. Mechanisms of acquired immunity are not at full strength until some time after birth. Most of the protection for a fetus or newborn comes from maternal IgG that crosses the placenta during pregnancy. Only IgG can cross the placenta because only the g-chain CH1 and CH3 domains can bind to placental cells. Intact IgG or Fc fragments from IgG can cross the placenta, but Fab or F(ab9)2 cannot. Maternal IgA, secreted in breast milk, neutralizes pathogens in the infant’s gut. The third area of biologic activity is the binding of IgE to mast cells and basophil receptors through theirFc regions. Because of this “stickiness,” IgE antibodies are called cytophilic antibodies. The reaction following the second exposure of specific antigen with IgE molecules bound to mast cells triggers allergic responses. Lymphocytes have Fc receptors for IgG that regulate IgG–antigen complex-mediated antibody feedback. MONOCLONAL ANTIBODIES ARE PUREANTIBODIES WITH SINGLE ANTIGENICDETERMINANT SPECIFICITIES Normal serum contains 1016 antibody molecules per milliliter. These antibodies can be collected from experimental animals and have long been an important tool of investigators, who have used them to identify or label molecules or cells and to separate molecules or cells from mixtures. A concern, however, has always been the variability of antisera The development of hybridization techniques allowed for the production of one kind of specific antibody by immortalized antibody-producing cells. Methods were then developed to screen for and produce large numbers of these antibodies for use in many applications. Antibodies of a single idiotype produced by immortalized B cells are called monoclonal antibodies. Regrettably, normal antibody-secreting cells are end cells of a differentiation series; thus they cannot be maintained in culture. In contrast, myeloma cells are immortal. Therefore: “Why not use immunoglobulins produced by myeloma cells?” The reason myeloma immunoglobulins cannot be used is twofold: (1) their antigen specificity is usually unknown and (2) it is difficult to tailor- make antigen-specific myelomas. The second problem is overshadowed by the question “Is the combining site of the myeloma protein an accurate representation of the antibody produced during an immune response?” What would happen if one combined the characteristics of each cell into one? Georges Kohler and Cesar Milstein fused cells secreting antibody of one specificity with myeloma cells. The resulting clone of cells is a hybrid-myeloma or a hybridoma. The hybridoma cells inherit the lymphocyte’s property of specific-antibody production and the immortality of the myeloma cell. In 1975, Kohler and Milstein published a short paper in Nature detailing how continuous cultures of fused cells secreting a monoclonal antibody of predefined specificity were produced. They were awarded the 1984 Nobel Prize in Physiology and Medicine for their work. Monoclonal Antibodies Have Many Applications Monoclonal antibodies ushered in a new era in immunologic research and in the application of immunologic assays to basic and clinical questions. The impact of hybridoma technology is obvious
- 6. when we begin to look at the many situations in which it has been successfully used Monoclonal antibodies are not without problems (1) the affinity for antigen may be low, (2) individual species of monoclonal antibodies do not readily activate complement or precipitate or agglutinate antigens in vitro, and (3) monoclonals are difficult to use in vivo in humans because of the difficulty of producing human, as opposed to mouse, hybridomas. To avoid these problems, antibody–gene DNA that has been manipulated in vitro is introduced into myeloma cells. The process is called transfection, and the resulting transfected cells are called transfectomas. Transfection is the integration of donor DNA into a cell’s chromosomes If the recipient cell lacks the genetic trait encoded by the donor DNA, the recipient cell acquires that trait.8 Transfection uses several recombinant DNA techniques to produce an entire new family of novel, tailor-made monoclonal antibodies called either hybrid, chimeric, or recombinant, monoclonal antibodies. The goal is to humanize antibody. Custom-made antibodies can be molecules with variable regions joined to different isotypic constant regions. These antibodies could enhance the binding of antigen-specific antibodies to protein A (a cell wall component of staphylococci that binds specifically to the Fc portion of IgG molecules) by changing the Fc part, or they could change the antibody’s ability to bind complement (keep the same antigen reactivity but change effector function). Custom-made antibodies also can be generated that have unique heavy- and light-chain combinations. This allows one antibody molecule to bind two different antigenic determinants. Furthermore, molecules with Fab antibody sequences can be fused with nonantibody sequences (such as enzyme or toxin sequences). This approach could make chimeric antibodies that are part antibody and part enzyme (which has use in immunoassays) or part toxin (antibody directs toxin to a specific site). The applications of these recombinant antibodies seem limitless. Why not bypass hybridoma technology and even immunization? You can. Lymphocyte V-region gene repertoires are harvested or assembled in vitro and cloned for display of heavy- and light-chain Fab fragments on the surface of bacteriophage, thus the name phage display antibodies. Rare phage display antibody is selected by binding to antigen, and soluble Fab fragments are expressed from infected bacteria. Ironically, we are using the bacteria that our immune system is designed to defend against to make antibodies.
- 7. Types of Antigens T-dependent Polysaccharides Properties Polymeric structure Polyclonal B cell activation Yes -Type 1 (TI-1) No - Type 2 (TI-2) Resistance to degradation
- 8. Prepared By Amjad Khan Program Microbiology