Immunology Unit 2 PDF - Chapter 11: Antigens

## Chapter-11: Antigens ### Learning Outcomes After reading this chapter the learner would be able to understand and appreciate the followings: * Antigens and immunogens * Chemical nature of antigens * Structure of antigen * Types of antigens * Superantigens * Antigenicity and immunogenicity * Ha...

## Chapter-11: Antigens ### Learning Outcomes After reading this chapter the learner would be able to understand and appreciate the followings: * Antigens and immunogens * Chemical nature of antigens * Structure of antigen * Types of antigens * Superantigens * Antigenicity and immunogenicity * Haptens and their role in diagnosis * Factors influencing immunogenicity * Adjuvants * Epitopes (antigenic determinants) and their types * B cell epitopes and T cell epitopes ### Definitions #### A. Antigens Antigens (Anti body generator) are the foreign substances or molecules which, when enters the body, trigger the generation of antibodies. Antigen (Ag) can be defined as any substance or molecule that can bind to specific antibody (Ab) or specific antigen receptors on lymphocytes. #### B. Immunogens In immunology, though the terms "immunogens" and "antigens" are synonymously used, there is an indirect difference between the two. Any substance or molecule that induces a specific immune response by activating lymphocytes (B cells or T cells or both) is called an immunogen (immunity generator). However, a substance or molecule that reacts with the products of a specific immune response is called antigen. Therefore, all immunogens are antigens, but not all antigens are immunogens. For example, hapten is a small-molecule antigen, which by itself does not induce a specific immune response (i.e. they lack immunogenicity), can stimulate an immune response if covalently coupled to a suitable carrier. Although all antigens are recognized by specific lymphocytes or by antibodies, only some antigens are capable of activating lymphocytes. Those antigens that are capable of inducing an immune response are called immunogens. Hence inmmunogens are immunogenic antigens. Immunogen is an antigen substance (adduct) that is able to trigger a humoral (innate) or cell-mediated immune response. ### Chemical Nature of Antigens (Immunogens) Antigens or immunogens include a variety of molecules such as proteins, polysaccharides, phospholipids, nucleic acids. Even simple intermediary metabolites, sugars, lipids, hormones and autacoids can be antigens. Bacteria, viruses, fungi, protozoa, worms, transplanted tissues, transfused blood cells and pollens display, produce or release nonself substances which are called antigens. Some "self" substances can be antigenic such as tumor antigens. Antigens are usually carried by proteins and polysaccharides, and less frequently, lipids. Lipids and nucleic acids are antigenic only when combined with proteins and polysaccharides. * **Proteins:** The vast majority of proteins are good immunogens due to their large molecular size, heterogeneity and complexity. Immunogenic proteins may be pure protein or glycoproteins or lipoproteins. * **Polysaccharides:** Pure polysaccharides and lipoproteins are good immunogens. * **Nucleic acids:** Nucleic acids are usually poorly immunogenic. However, they may become immunogenic when single stranded or when complexed with proteins. * **Lipids:** In general lipids are non-immunogenic, although they may be haptens. ### Structure of Antigen (Epitopes or Antigenic Determinants) The entire immunogen or antigen molecule does not interact with antibody or immune cells. But the specific regions/chemical groups of antigen molecule that can bind specifically to paratope (antigen-binding site) of antibody molecules or T-cell receptors are called epitopes or determinants or antigenic determinants. Naturally occurring epitopes can be 4-6 amino acid or sugar residues long. ### Functions of Antigens * Elicit both cell-mediated and humoral immune responses. * They bring about allograft rejection. * The determine blood groups. ### Types of Antigens * **On the basis of immune response, antigens are of two types:** * **Complete antigens or immunogen:** These are the substances of high molecular weight (more than 10,000) which are able to generate immune response by themselves. They are chemically proteins or polysaccharides. * **Incomplete antigen or hapten** * **On the basis of origin, antigens are classified into following types** * **Exogenous antigens** * **Autoantigens** * **Tumor (cancer) antigens** * **T cell independent antigens** * **Superantigens** * **Cross-reactive antigens** * **Endogenous antigens** * **Alloantigens** * **Neoantigens** * **T cell dependent antigens** * **Blood antigens** #### 1. Exogenous antigens: These are the foreign or non-self antigens that enter into the body from outside, for example by inhalation, ingestion or injection. They can be microorganisms like bamcteria, viruses, protozoa, fungi, worms, allergens, transfused blood cells etc. #### 2. Endogenous antigens: These are the native or self-antigens which generated by the own body cells due to abnormal physiology or infection. The endogenous antigens can be autoantigens and alloantigens. #### 3. Autoantigens: An autoantigen is usually a normal protein or complex of proteins (and sometimes nucleic acids or nucleoproteins, thyroglobulin) that is recognized by the immune system of patients suffering from specific autoimmune diseases. Under normal conditions, autoantigens are not targeted by immune system, but genetic and environmental factors may cause the breakdown of the normal immunological tolerance for such antigen in these patients and their immune system recognizes as nonself antigen. Some auto-antigens (antigens of eye lens, cornea, spermatozoa, skin, lungs, kidney) which do not, under normal conditions, come in contact with the host immune system- then antibodies are not produced against such cells and tissues. The antibodies generated against autoantigens causes the development of corneal opacity, orchitis, glomerulonephritis, intestinal thyroiditis etc. #### 4. Alloantigens: An alloantigen is a genetically determined antigen, such as histocompatibility or red blood cell antigens, that is present in some but not all individuals of a species and capable of inducing the production of an alloantibody by individuals which lack it. It is also called isoantigen. #### 5. Tumor antigen: Tumor antigen is a protein or any molecule produced during mutation or tumor development in a normal cell. These molecules or proteins which are not previously exposed to the immune system act as non-self and presented by MHC class I or MHC class II molecules on the surface of the cell to recognize by T cells. After recognition by T cell it triggers immune response in the host against that particular protein. Cytotoxic T lymphocytes that recognize antigens through MHC class I molecule may destroy the tumor cells before they proliferate or metastasize. Tumor antigens can also be present on the surface of the tumor in the form of a mutated receptor and recognized by B cells. Normal proteins in the body are not antigenic because of self-tolerance, a process in which self-reacting cytotoxic T lymphocytes (CTLs) and autoantibody-producing B lymphocytes are gather in thymus and bone marrow respectively. Sometime, tumor antigens are also called as neoantigens. These antigens are presented by tumor cells and never by the normal cells and therefore called as tumor-specific antigens (TSAs). Some important tumor antigens are alphafetoprotein (AFP), carcinoembryonic antigen (CEA), epithelial tumor antigen (ETA), Melanoma-associated antigen (MAGE). #### 6. Neoantigen: It is the newly formed antigen that has not been previously recognized by the immune system. Neoantigens are often associated with tumor antigens and are found in oncogenic cells. #### 7. T cell independent antigens: The antigens which can directly stimulate the B cells to produce antibody without the T cells help are called “T cell independent antigens”. In general, polysaccharides are T-independent antigens. The T cell independent antigens show following properties: * **Polymeric structure:** These antigens have the same epitopes repeating many times. * **Resistance to degradation:** The polysaccharides are generally more resistant to degradation and thus they persist for longer periods of time and continue to stimulate the immune system. * **Polyclonal activation of B cells:** Many of these antigens can activate B cell clones specific for other antigens (polyclonal activation). T-independent antigens can be subdivided into Type 1 and Type 2 based on their ability to polyclonally activate B cells. Type 1 T-independent antigens are polyclonal activators while Type 2 are not. * **Initiate humoral immune response:** The T-independent antigens bind to Ig receptors of B cells and bring about humoural immune response by producing only IgM type of antibodies. * **Produce weaker or no memory cells.** #### 8. T cell dependent antigens: T-dependent antigens are those that require the help of T cells to initiate immune response and antibody production. Hence, these antigens do not directly stimulate the production of antibody without the help of T cells. Proteins are T-dependent antigens. Structurally these antigens are characterized by a few copies of many different antigenic determinants. The T-dependent protein antigens are degraded by APC (antigen presenting cells) into smaller peptide fragments. The peptide fragments form a complex with MHC and then presented on the surface of APC as peptide-MHC complex. The T cells recognize the peptide-MHC complex. ### Superantigens Superantigens (SAgs) are microbial proteins that cause massive non-specific activation of T cells resulting in poly-clonal T cell activation. Compared to normal antigen-induced T cell response where 0.0001-0.001% of the body's T cells are activated; these SAgs are capable of activating up to 2-5% of the body's T cells. Conventional antigens are capable of activating a specific clone of T cells. In contrast, SAgs are capable of activating a number of T cell clones of different antigen specificities and thus are capable of eliciting a strong immune response. They are called superantigens because they resemble antigens in their binding to TCRs (T-cell receptors) and to class II MHC molecules but activate many more T cells than do conventional peptide antigens. Superantigens can be of following types: (a) Exogenous SAgs includes soluble exotoxins (proteins) secreted by Straphylococcus and other bacteria. (b) Endogenous SAgs are virus encoded cell membrane proteins. For example, the endogenous Sags of mouse mammary tumor virus (MMTV) causes a deletion of T cells bearing a particular Vẞ element, amplification of infected B cells and transmission of virus to the offspring. (c) B-cell SAgs that stimulate predominantly B cells. Superantigens are not processed by antigen presenting cells (APCs), instead bind directly to class II MHC molecules expressed on APC surface. Moreover, SAgs allow cross-link class II MHC molecules on APC surface and also simultaneously cross-link class II MHC molecule to TCR. They can also bind directly to the Vẞ region of TCR outside the usual antigen binding groove. SAgs do not react with CDRs (complementarity-determining regions), but rather bind to consensus regions in the FRs (framework regions). SAgs cause strong T cell response, even in the absence of co-stimulations. Such binding ability of SAgs result in the activation of multiple clone of T cells non-specifically, thus eliciting a heightened immune response. They can simultaneously bind and activate class II MHC molecule on APCs outside the antigen binding cleft and activate T-cells bearing a particular T-cell receptor with Vẞ regions. SAgs activate T cells leading to the release of large amount of cytokines. Sometimes induce massive cytokine release (cytokine storm) that can ultimately result in deletion of many T cells, hypertension, fever, food poisoning, shock, organ failure and death. The release of excess cytokines, such as IFN-y, strongly activates macrophages, sometimes lead to septic shock and other complications. ### Antigencity and Immunogenicity Any substance/molecule that induces a specific immune response (humoral and/or cell-mediated) is called an antigen and more specifically immunogen. The ability of an immunogen to induce an immune response (humoral and/or cell-mediated) is called immunogenicity. The products of immune response can be antibodies (in humoral immune response), or cytokines and cytotoxic factors (in cell-mediated immune response). Antigenicity is the ability to combine specifically with the products of immunogenic responses such as and/or surface receptors on T cells. All immunogens have the property of immunogenicity as well as antigenicity, but reverse is not true. Therefore, all immunogens are antigens, but all antigens are not immunogens. For example, haptens are small-molecules (e.g., dinitrophenol, DNP) that are antigens (are antigenic) but not immunogenic. This is because haptens cannot induce immune response as such. Haptens only when conjugated with a large molecule can induce immune response and then can be immunogenic. ### Haptens Haptens are small molecules which could never induce immune response when administered by themselves, but when coupled with a carrier molecule such as protein, it can become immunogenic. Therefore, hapten is antigen but not an immunogen (cannot induce immune response); while hapten-carrier conjugate is both immunogen as well as antigen. Many biologically important agents such as drugs, fat-soluble vitamins (e.g. Vit.-E), steroids (lipids) and peptide hormones can functions as haptens. These haptens in conjugation with large proteins can be immunized to generate hapten-specific antibodies. These antibodies can be used to detect the presence of various chemicals in the body. Animal immunized with hapten-carrier conjugate can produce 3 types of antigenic determinants: (i) anti-hapten antibodies, (ii) antibodies against carrier and (iii) antibodies against new epitopes formed by conjugation of both hapten and carrier. Landsteiner used haptendinitrophenol (DNP) and carrier protein bovine serum albumin (BSA) and prepared the hapten-carrier conjugate i.e. DNP-BSA. When he injected/immunized the DNP-BSA conjugate to rabbit, he obtains three types of antibodies. ### Role of haptens in diagnosis * **In pregnancy test kit anti-hapten antibodies are used to detect the presence of HCG in woman's urine as a sign of pregnancy** * **Antibody-based immunoassays are now routinely used to detect and monitor the levels of specific chemicals in blood of patients. For example, antileukotriene C4 (anti-lipid antibodies) is used to assay the level of leukotriene C4 in blood in asthma patients. Further, after successful grafting, the levels of immuno-suppressive drugs (e.g., steroid like prednisone) in blood can be maintained by immunoassays.** ### Factors Influencing Immunogenicity Immunogenicity is determined by various properties of immunogen as well as the biological system. They are: #### A. Properties of the immunogen: * **Molecular size** * **Chemical composition and heterogeneity** * **Foreignness** * **Susceptibility to antigen processing and presentation with MHC molecule** #### B. Properties of the biological system: * **Host genetic makeup** * **Dosage and rout of immunogen administration** * **Use of adjuvants to enhance immunogenicity** #### 1. Molecular size: The molecular mass (size) of a macromolecule determines the immunogenicity. Generally substances with a molecular mass less than 5000-10000 Daltons (Da) are pore immunogens. The substances exceeding 10000 Da are most active immunogens. #### 2. Chemical composition and heterogeneity: The chemical complexity of a molecule affects immunogenicity. The synthetic homopolymers nonimmunogenic, irrespective of their size. Therefore, immunogens are usually heteropolymers (composed of different types of amino acids or sugars), which are influenced by molecular weight. Further, levels of organization - primary, secondary, tertiary and quaternary- decides the structural complexity of a protein and thus contribute to the immunogenicity. Macromolecules that cannot be degraded and presented with MHC molecules are poor immunogens, because the degradative enzymes within APCs can degrade only proteins contain L-amino acids, polymers of D-amino acids cannot be processed. Large molecules are more readily phagocytosed and processed, so they are more immunogenic than smaller molecules. Haptens like lipid antigens complexed with carrier proteins (e.g., BSA, or KLH/keyhole limpet hemocyanin protein) and immunized as lipid-protein conjugates can induce B cells releasing specific antibodies against lipids. #### 3. Foreignness: Host immune system can distinguish self from nonself and is usually tolerant (nonresponsive) to self-antigens. In order to elicit an immune response, a molecule must be recognized as non-self (foreign). If a molecule has not been exposed to immature lymphocytes, it may be later recognized as non-self or foreign. The foreignness of a molecule depends on the degree to which it has been exposed to immature lymphocytes. For example, self-components like sperm and corneal cells which remain away from the reach of immune system, when injected into the same animal, they can elicit immune response. Greater the phylogenetic distance between two species greater will be the structural (and therefore the antigenic) disparity between them. For example, the BSA (Bovine serum albumin) would be more immunogenic in a rabbit than when injected into a cow, because of disparity between the two species. But exceptionally, macromolecules like collagen and cytochrome c remain the highly conserved during the evolutionary process exhibit little immunogenicity across various species. #### 4. Genetic variability: The genetic makeup of an immunized organism can influences the degree and type of immune response. For example, the MHC genes encoding MHC molecules, the genes encoding B cell receptors and T cell receptors are important genetic properties of the biological system that determines the immunogenicity. Experimental findings suggests that MHC genes show high degree of polymorphism and therefore, the MHC molecules needed for the presentation of processed antigen to T cells determine the degree of immune responsiveness. #### 5. Antigen dose and rout of immunization: Activation of lymphocytes depends upon the dose and the rout of administration of an immunogen. ### Adjuvants Adjuvants (L. adjuvare=to help) are the compounds that boost the immune response when mixed and administered with antigens. #### Examples of Adjuvants: * Micrbial products such as bacterial LPS (lipopoly saccharides) and killed Mycobacteria. * Synthetic polyribonucleotides * Aluminum potassium sulfate (alum) * Freund's incomplete adjuvants (water-in oil emulsions without killed Mycobacterium tuberculosis) * Freund's complete adjuvants (water-in-oil emulsions with killed Mycobacterium tuberculosis) * Synthetic Toll-like receptor (TLR) activators such as monophosphoryl lipid A (MPL) and CpG motifs, recombinant cytokines like IL-1, IL-12 and IFN-y. These are safe for human use. * Plasmid DNA has intrinsic adjuvant-like activities. #### Adjuvants exert one or more of the following effects: * **Prolong the persistence of antigens:** For example, in mice, Freund's incomplete adjuvant can prolong the persistence of antigen by slowly releasing the antigen from the site of injection. * **Enhance immune responses:** Adjuvants facilitate immune responses by enhancing the expression of high levels of costimulators, MHC molecules on APCs as well as the antigen presenting function of APCs. Adjuvants also induce the release of cytokines. For example, Freund's complete adjuvant (powerful adjuvant than the incomplete adjuvant) when given in experimental animals like mice, increase the phagocytic activity of dendritic cells and macrophages and induce higher expression of costimulators and production of cytokines that enhance T cell growth and differentiation. * **Adjuvants stimulate the non-specific proliferation of lymphocytes.** * **Adjuvants stimulate local inflammatory response:** Freund's adjuvants and alum can induce local inflammatory response leading to granuloma formation. These adjuvants attract macrophages and lymphocytes at the site of injection. Sometimes a macrophage-rich dense mass develop called granuloma.. * **Adjuvants induce enhanced primary T cell response to purified protein antigens in vaccines by stimulating innate immune responses at the site of antigen exposure.** ### Epitopes (Antigenic Determinants) Epitopes or antigenic determinants are the sites on the immunogen molecule (macromolecule) to which any antibody, B cell or T cell can specifically bind. Macromolecules generally contain multiple epitopes and each epitope can bind by an antibody. The number of epitopes in an antigen is called valency of antigen. The antigen with multiple epitopes are called polyvalent or multivalent antigen, e.g., polysaccharides and nucleic acids. Microbial cell surface often display multiple identical epitopes of protein or carbohydrate. Large proteins exhibit different epitopes hence, are not polyvalent. In contrast to complete antigens, haptens contain a single epitope. Epitopes, on the basis of position on the antigen, can be surface epitopes or hidden epitopes. The surface epitopes are functional and accessible to antigens and hence are called accessible epitopes, while the hidden epitopes are non-functional and are not accessible for binding unless they are exposed are called inaccessible epitopes. ### Epitopes are of following types: * **Linear or sequential determinants (epitopes)**: An epitope consists of adjacent 6-8 amino acid residues are called linear epitope. The linear epitopes are accessible to antibodies when located on external surface. Native proteins when denatured may expose hidden linear determinants. * **Conformational or non sequential determinants (epitopes):** These are the epitopes formed by spatial folding of proteins that bring some distantly placed amino acid residues closer. * **Neoantigenic determinants/epitopes:** Sometimes covalent modifications (phosphorylation, glycosylation, acetylation or proteolysis) of proteins may generate new epitopes in proteins, and such newly produced epitopes which did not exist earlier are called as neoantigenic epitopes. ### B-CELL EPITOPES & T-CELL EPITOPES The recognition of antigens (epitopes) by B cells and T cells is fundamentally different. Mice immunized with human glucagon (29 amino acid residue long) revealed that antibodies produced by B cells bind to the epitopes in N-terminal portion, while the T cells bind to the epitopes in C-terminal portion of the glucagon. B cells recognize soluble antigens. The epitopes to which B cells can bind by their surface receptors is called B-cell epitopes. The B-cell epitopes have following properties: * They should be highly accessible sites present on the exposed surface of the immunogen (antigen). * On native protein antigens, the B-cell epitopes are generally protruding regions and composed of hydrophilic amino acids. The hidden epitopes consisting of hydrophobic amino acids cannot function as B-cell epitope. * They can be linear epitopes (sequential contiguous residues) or conformational epitopes (nonsequential residues brought together by folded conformation) * They must have complementary shape to the paratope (antibody's binding site). * Rigid B-cell epitopes bind less effectively with antibody. Tainer et al analyzed the positions of B-cell epitopes on many protein antigens (m.yoglobin, hemoglobin, cytochrome c etc.) and observed that the major B-cell epitopes are located in the most mobile regions of these proteins. From these observations, they proposed that site mobility of B-cell epitopes located in flexible regions of an immunogen can maximize the complementarity with paratopes. * In complex proteins like mammalian albumins and BSA, B-cell epitopes are found to be overlapping. In case of overlapping, the same B-cell epitope can be recognized by different antibodies. For example, in BSA (Bovine serum albumin) about 25 different overlapping B-cell epitopes found which can be recognized by 64 different antibodies. * Within an animal, certain B-cell epitopes are more immunogenic than others. Such epitopes are called immuno-dominant, which can induce a more pronounced immune response in an organism than other epitopes. The epitopes to which T cells can bind by their surface receptors (TCRs) is called T-cell epitopes. The T-cell epitopes have following properties: * They are internal linear peptides of about 7 amino acid residues long). * They are generated by processing of antigens in APCs. * They interact specifically with MHC (major histocompatibility) molecules, expressed on the surface of APCs. The peptide-MHC complex when presented on the cell surface is recognized by T cells and induces immune response. * When they are bound to Class I MHC molecules are found on every nucleated cell of the body while class II MHC molecules express on professional APCs such as dendritic cells, macrophages and B cells. MHC class I molecules usually present peptides between 8-11 amino acids in length, whereas the peptides binding to MHC class II molecules may have length from 12-25 amino acids derived through the proteolysis of pathogen antigens, and present them on cell surface for recognition by CD4+ T cells. * All T-cell epitopes are good MHC binders, but not all good MHC binders are T-cell epitopes. ### Summary * The terms immunogens and antigens are synonymously used. * Immunogens are substances or molecules that can mobilize adaptive defenses by activating lymphocytes (B cells and T cells) and provoke an immune response. * Antigen is a molecule that reacts with the products of a specific immune response. * Antigen is contraction of "antibody generator". * Complete antigens have two important functional properties: immunogenicity (ability to stimulate specific lymphocytes to multiply) and reactivity (ability to react with the activated lymphocytes and the antibodies released by immunogenic reactions). * In complete antigens are called haptens. Unless attached to protein carriers, haptens have reactivity but not immunogenicity. * Epitopes or antigenic determinants are the sites on the immunogen / antigen molecule to which any antibody, B-cell or T cell can bound. * Most antigens (both natural and synthetic) that are not normally present in the body are treated as non-self (or intruders), Self-antigens particularly МНС proteins mark a cell as self. MHC proteins are a group of cell surface glycoproteins. T lymphocytes can only bind antigens that are presented to them on MHC proteins. * Adjuvants are the compounds that boost immune response when mixed and administered with antigens. ### Model Questions * **Very Short Answer Type Questions** * Define antigens. * Antigens are specific chemicals that can mobilize the adaptive defenses and provoke an immune response. * What are the chemical natures of antigens? * Antigens are usually proteins, but some lipids, polysaccharides, and nucleic acids are also antigens. * What is an antigenic determinant? * Antigenic determinant (or epitope) is the smallest unit of antigen that is capable of binding with paratope of antibodies. * What is adjuvant? * An adjuvant is a substance that when mixed with an immunogen and injected with it, enhances (boost) the immune response against the immunogen. * Why invertebrates lack adaptive immunity? * Because invertebrates do not have lymphocytes. * **Fill in the Blanks** * Antigen is a contraction of _________ * Antibody-generator * The epitope of antigen bound to __________ of antibody. * Paratope * When one epitope is present, the antigen is called __________, while the antigen with multiple copy of the same epitope is called __________. * Monovalent; Polyvalent * The antigen receptor on B lymphocytes is called __________. * B-cell receptor (BCR) * T-cell antigen receptor (TCR) recognize antigen when they are associated with surface bound __________ proteins. * MHC * **Short Answer Type Questions. Answer in two or three sentences** * What are the two important properties of complete antigens? * Two important properties of complete antigens are: (a) Immunogenicity- which is the ability to stimulate specific lymphocytes to proliferate (multiply), and (b) Reactivity- the ability to react with the activated lymphocytes and the antibody released by immunogenic reactions. * Why haptens are incomplete antigens? * Haptens are small molecules that fail to induce immune responses of their own, unless attached to protein carriers. * Differentiate between affinity and aviditry with reference to antigen. * Affinity is the strength with which a monovalent antigen (paratope) of antibody, whereas avidity is the strength of antigen-antibody binding when multiple epitopes on an antigen interact with multiple binding sites of an antibody. * What makes a cell as "self" as opposed to "non-self"? * Self-antigents particularly MHC proteins, mark a cell as self. * **Short Answer Type Questions. Answer within 75 words** * **Long Answer Type Questions** ## Chapter-12: Immunoglobulins ### Learning Outcomes After reading this chapter the learner would be able to understand and appreciate the followings: * Various antigen-binding molecules * Immunoglobulins- Definition, occurrence and forms. * Basic structure of immunoglobulins * Fragments of immunoglobulins * Different classes of immunoglobulins * Immunoglobulin G * Immunoglobulin A * Immunoglobulin M * Immunoglobulin D * Immunoglobulin E * Abzymes (Catalytic antibodies) ### Introduction There are three types of antigen-binding molecules known to be used by the adaptive immune system. They are: * Immunoglobulins (Igs) * T-cell receptors (TCRs) * Major histocompatibility complex (MHC) molecules Of these, the Igs are the first discovered antigen-binding molecules that can recognize and bind widest range of antigens with greatest affinity. ### Definitions The immunoglobulins (Igs) are the heterogeneous population of antibodies (Abs) that interact specifically with the antigens (Ags) that elicited their formation. Igs are the gamma-globulin class of serum proteins that take part in humoral immune response. The terms immunoglobulins and antibodies are being interchangeably used. ### Occurrence Immunoglobulins are glycoprotein molecules present in highest concentration in plasma or serum. Igs constitute 20% of the total plasma proteins by weight. They can also found in various body fluids including tears, milk, urine, nasal mucus, sweat etc. ### Forms of Immunoglobulins B lymphocytes (B cells) of vertebrates are the only cells that synthesize Igs. They express immunoglobulins in two forms, namely: * **Membrane-bound immunoglobulins (mIg):** The naïve (newly formed or virgin) B cells express mIg which are attached to the surface of B-cells and act as antigen-receptors or B cell receptors (BCR). Each B cells has about 105 such antibodies in its membrane surface. Initially B cell expresses IgM and IgD on the surface. * **Secreted immunoglobulins (sIg):** They are the circulating soluble forms of Igs found in plasma, serum and tissue fluids. When a naïve or memory B cell binds with mIg it initiates cell proliferation and differentiation to produce either a clone of plasma cell or memory cells. Each clone of plasma cells (late stage of activated B cells) secretes homogeneous antibodies (only one type of Igs) which have the same specificity for antigen that is identical to the original surface receptor of B cells. ### Basic Structure of Immunoglobulins Based on the experimental data of proteolytic cleavage of IgG, Rodney Porter (1962) proposed a basic structure for immunoglobulin which is common to all classes of Igs. The basic structural unit of Ig is a Y-shaped or T-shaped molecule consists of a tail (stalk) and two arms (limbs) joined by a flexible hinge region. * **Tail:** The tail of the Ig mediates a variety of biological activities such as activating complement system and phagocytic cells. * **Arms:** The tip of each arm of Ig contains the antigen-binding site (paratope). * **Hinge:** The flexible hinge of Ig allows the distance of two arms and increases the efficiency of antigen binding and cross-linking. The Gerald Edelman (1960) and Rodney Porter (1962) found that the basic unit of Ig consists of 4 polypeptide chains i.e. 2 identical light (L) chains and 2 identical heavy (H) chains held together by disulfide bonds. This basic monomeric heterodimer structure (H2L2) is common in all classes of Igs. ### Immunoglobulin Domains The basic four-chain structure is functional at quaternary level. Each of the L- and H-chains is folded into a tertiary structure composed of repeating homologous globular domains. Adjacent domains are interconnected by stretches of polypeptide chains. Each domain is 110 amino acid residues long with an intrachain disulfide bond that forms a loop of 60-70 amino acid residues. Each L chain consists of one variable (VL) and one constant (CL) domain, while most H chains consists of one variable domain (VH) and three or four constant domains (CH1, CH2, CH3 and CH4) based on antibody class. The CH2 domains internally attached to oligosaccharide chains (glycosylation). The variable domains of adjacent H and L chains come together to form antigen-binding site, while the constant domains (except CH1) form the Fc region that perform a number of biological effector function. X-ray crystallographic studies revealed that each Ig domain folds independently to form a compact functional unit called the immunoglobulin fold. This structure composed of a "sandwich" of two ẞ pleated sheets (one layer composed of 3 strands of polypeptide chains and the other contains four strands). #### Light chains: * Each L chain has a molecular weight of ~ 25 kDa and consists of 214 amino acid residues. * Each L chain has two repeating globular/spherical folds called domains. An Ig domain consists of 110 residues with an intrachain disulfide bond. In each domain, the intrachain disulfide bond forms a loop of 60-70 amino acid residues. * The L chain has amino (NH2) terminal end and carboxy (COOH) terminal end. #### Heavy chains: * Each H chain has a molecular weight of ~ 55 kDa and consists of 446-700 amino acid residues. * Each H chain has four Ig domains one variable domain (VH) towards N-terminal and three constant domains (CH1, CH2 and CH3) towards C-terminal. * Like L chain, each domain of H chain consists of 110 amino acid residues with an intrachin disulfide bond forms a loop of 60-70 amino acid residues. * On the basis of different sequence of constant regions, H chains are of 5 different types, designated by Greek letters namely: * Alpha (a) chain in IgA * Delta (8) chain in IgD * Epsilon (8) chain in IgE * Gamma (y) chain in IgG * Mu (u) chain in IgM ### Antigen-binding site (=Paratope) It is now clear that about 20-30 amino acid residues in the variable region of L and H chains form the antigen binding site. The amino acid sequence data revealed that VL and VH domains are not uniformly variable. Rather, each variable domain consists of three small hypervariable regions (HVRs) and four relatively constant framework regions (FRs). The X-ray diffraction studies indicate that the 3 HVRs arranged in form of 3 hypervariable loops that connect the ẞ-strand of VH and VL domains. Each HVR consists of 5-10 amino acids. In 1970, Elvin Kabat proposed that the 3 HVRs of VL domain and 3 HVRs of VH domains are brought together to form the antigen-binding site and the nature of amino acid residues determine the binding specificity for antibody. Since the antigen-binding site is complimentary to the 3-D structure of the epitope, the HVRs are also called as complementarity-determining regions (CDRs). Thus, both VL and VH domains has 3 CDRs each i.e. CDR1, CDR2 and CDR3. ### Fragments of Immunoglobulins #### (i) Limited proteolysis with enzyme papain Porter (mid-1950s) when treated IgG with proteolytic enzyme papain, the IgG cleaved into three fragments: two identical Fab fragments and the third fragment called Fc fragment. * **Fab (fragment antigen-binding):** Each Fab fragment has single antigen-binding site (paratope) at one end. The part of H chain present in the Fab is known as Fd piece. * **Fc (fragment crystallizable):** It lacks the ability to bind with antigen. It is named so because it readily crystallizes in cold storage. The Fc fragment found to contain carbohydrate and mediate the following functions: * Binds to complement component (C1q) to initiate the classical pathway complement cascade. * Binds to cell receptors i.e. Fc receptors (FcRs) on phagocytic cells, NK cells and mast cells. * Involved in degranulation of mast cells. * Determines the secretory ability of Ig molecules. * Determines the passage of Ig across placenta. * Antigenic determinants that distinguish one class of antibody from another are also located on Fc. #### (ii) Limited proteolysis with enzyme pepsin Nisonoff when treated IgG with proteolytic enzyme pepsin, the IgG is cleaved into a large fragment designated as F(ab')2 fragment which consists of two covalently linked F(ab') fragment (each slightly larger than Fab fragment); the remaining Fc fragment is quickly degraded by pepsin into numerous small peptides and leaves a dimer of C-terminal quarter called pFc fragment. #### (iii) Limited proteolysis with reducing agents Gerald Edelman (1960) when treated IgG with a reducing agent such as mercaptoethanol, in the presence of urea, the