IMMUNOLOGY AND MEDICAL MICROBIOLOGY

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1 CONTENTS IMMUNOLOGY AND MEDICAL MICROBIOLOGY Antibodies Ajit Singh Associate Professor I/c Immunology Section Department of Veterinary Microbiology (ICAR Centre of Advanced Studies) College of Veterinary Sciences CCS Haryana Agricultural University Hisar (Haryana). 23- Jan-2006 (Revised 03- Jul-2006) What are Antibodies? B lymphocyte clones differentiate into plasma cells that secrete Abs Abs are heterogeneous glycoproteins that share many common physico-chemical and structural features Shapes of antigen-binding site/ paratope Dissecting the structure of Abs in order to understand their functions Antibodies vis-a-vis enzymes Different Ab classes and subclasses are for diverse effector functions Structural variants of Abs are genetically determined and antigenically distinguishable Biological functions of Abs Abs as biocatalysts Forces involved in antibody-antigen interactions Affinity, avidity and specificity of antibodies Ig genes and generation of antibody diversity Major genetic mechanisms that diversify Ab specificities and effector functions Theories of antibody production - A brief account Immunotechnology for production of antibodies and the antigen-binder fragments Origin and Evolution of Igs Immunoglobulin superfamily (IgSF) Keywords Antibody structure, functions, classes, diversity, Ig superfamily, immunotechnology

2 What are Antibodies? When a vertebrate animal, including human is injected with or naturally exposed to certain foreign substances, called antigens, it produces specific humoral factors. These appear in peripheral blood after a few days in order to bind and eliminate the antigens from the body. These specific humoral (fluid-borne) factors that provide humoral immunity to the host against the offending antigens are collectively known as antibodies (Abs). Abs were first detected in the sera of animals injected with bacterial exotoxins, such as tetanus toxin and diphtheria toxin, by Emil von Behring and Shibasaburo Kitasato in the year 1886 A.D. Very vast information has accumulated since then on the biochemical nature, physico-chemical properties, biological functions, and diverse uses of Abs. This chapter aims at discussion of all such features, functions, and uses of Abs, the magic bullets as Paul Ehrlich called them at the dawn of the 19 th century. How studies on Abs progressed can be seen in distinct eras in the history of immunology (Table 1). Table 1: Eras in the progress of studies on Antibodies (Immunoglobulins) Era Time span Important events (the most prominent scientists involved) Early s Discovery of antitoxins in serum (E.A. von Behring & S. Kitasato, 1986) Abs in serodiagnosis & serotherapy Side-chain theory of Ab formation (P. Ehrlich, 1900) Immunochemistry s Specificity of Ab defined (K. Landsteiner, ) Ab chemistry & quantitative precipitation reaction (M. Heidelberger, 1920s) Physico-chemical characteristics of Abs (Marrack, 1930s) Ag template theory of Ab production (Horowitz, Alexander & Pauling, ) Isotypic, allotypic and idiotypic variants of Abs found (J. Oudin, ) Structure of Ab molecule elucidated (Edelman, Porter, Nisonoff & Kunkel, ) Immunobiology s Clonal selection theory of Ab production (FM Burnet, 1960) Ab producing B cells detected (Pernis, 1969) Network theory of immune system (NK Jerne, 1974) Modern to date Modern immunotechnology begins with introduction of hybridoma technique (G Kohler & C Milstein, 1975) Ig genes and genetic basis of Ab diversity elucidated (S Tonegawa, 1980) rdna based techniques for production of Abs and their Ag-binding fragments in various formats in E. coli, yeast, insect cells, mammalian cells, plants, transgenic animals and phage display system; newer roles of Abs being explored such as in catalysis, proteomics, biosensors, etc. 2

3 B lymphocyte clones differentiate into plasma cells that secrete Abs Abs are products of antigen-specific B lymphocyte clones that are responsible for adaptive humoral immunity in vertebrate hosts. They are produced mainly in secondary lymphoid organs and tissues, such as lymph nodes, spleen, mucosa-associated lymphoid tissues, etc. After stimulation by the antigen, specific B cell clones undergo multiple cycles of cell division and terminally differentiate into plasma cells that secrete specific Abs in large amounts in lymphoid tissues. From lymphoid tissues, Abs are disseminated via peripheral blood circulation to entire body, tissue fluids and secretions. Antibodies (Abs): Box 1: Antibodies as antigen-specific defence molecules Are humoral (fluid-borne) factors produced in vertebrates in response to foreign substances, known as antigens (Ags) Were discovered by E. von Behring & S. Kitasato in 1886 A.D. Are produced by Ag-specific B cell clones & secreted by Ag-specific plasma cells Are glycoproteins made of two identical light (L) chains (25-30 kda) and two identical heavy (H) chains (50-70 kda) Have the monomer structure: L-H-H-L, and the multimeric forms (L 2 H 2 ) 2-6 also exist Exist as classes & subclasses with diverse functions In addition to isotypic (classes & subclasses), have allotypic and idiotypic variants Have five different classes in humans: IgM, IgG, IgA, IgE and IgD Human IgG has four subclasses: IgG1, IgG2, IgG3 & IgG4 Abs are heterogeneous glycoproteins that share many common physico-chemical and structural features Abs are glycoproteins of heterogeneous size, charge, amino acid composition and antigenic nature. Each Ab is however basically made up of four polypeptide chains: two identical smaller polypeptide chains of about kda size and two identical bigger polypeptide chains of kda size. The smaller polypeptide chain is designated as L (light) chain and the bigger as H (heavy) chain. L chain is composed of about 220 amino acids (AAs), whereas H chains have about 440 or 550 AAs in them. Only the H chains are glycosylated. In the basic monomeric form of Ab, the polypeptide chains are joined together by interchain disulphide bonds between cysteine residues in a manner that a structure that may be written as [L-H-H-L] is obtained (Fig. 1). Certain Abs also exist as multimers of the basic monomeric form i.e., as (L 2 H 2 ) n. The value of n may vary from 2 to 6. Ab molecules migrate to the electrophoretic region of γ- and β- globulins of serum. As most Abs exhibit γ- electrophoretic mobility, they are called γ-globulins. Macroglobulins, i.e, the largest sized Abs (IgM) however, migrate to β region. Being involved in immune responses, γ- and β- 3

4 globulins collectively are called immunoglobulins (Igs). Monomeric Ab molecules have sedimentation coefficient of 7S- 8S, whereas di- and pentameric molecules have 11S and 19S, respectively. The comparison of amino acid sequences of Abs produced by different myelomas (cancer cells of B cell lineages producing homogeneous L or H chains or whole Ab molecules) or monoclonal Abs reveals that both L and H chains vary in their NH 2 - terminal (N-terminal) parts. The N- terminal half of L chain, which is about 110 AAs long and shows variations among L chains from different B cell clones is therefore called V L and the remaining COOH-terminal (Cterminal) part being almost invariable or constant among L chains from different B clones is called C L. Similarly, N-terminal one-fourth or one-fifth part of H chain, about 110 AAs, that shows variations among H chains from different B cell clones is called V H. The remaining C- terminal part, about 330 or 440 AAs being almost invariable or constant among H chains from different B clones is called C H (Fig. 1). However, two types of C L and at least eight types of C H exist in mice and humans, based on sequence differences. Physico-chemical properties of human Igs are presented in Table 2. 4

5 Table 2: Physico-chemical properties of human immunoglobulins Feature IgM Ig class IgGs IgAs (IgA1, (IgG1 to IgG4) IgA2), siga Size (kda) (IgG1, IgG2, IgG4) 170 (IgG3) 160 (IgAs) 385 (siga) IgE Sedimentation rate 19S 7S 7S (IgAs) 8S 7S 11S (siga) Structural formula (L 2 H 2 ) 5 -J L 2 H 2 (L 2 H 2 ) 2 L 2 H 2 L 2 H 2 (L 2 H 2 ) 2-J- SC (siga) L chain type κ / λ κ / λ κ / λ κ / λ κ / λ H chain type µ γ α ε δ Carbohydrate content 12% 2-3% 10% 10-12% 12% Electrophoretic β γ β γ β γ γ mobility Valency , 4 (siga) 2 2 Amino acid composition: L chain: H chain: J chain: SC chain: Ig domains: L chain: κ or λ 1V L +1C L H chain: 1V µ +4C µ Serum concentration mg/ml (siga) 550 (siga) 1V L +1C L 1V L +1C L 1V γ +3C γ 1V α +3C α mg/ml mg/ml (IgA1, IgA2) IgD 1V L +1C L 1V L +1C L 1V ε +4C ε 1V δ +3C δ 50 mg/ml 30 µg/ml Closer look into the sequences of V L and V H segments of several different Ab molecules reveals that each segment shows three hypervariable regions, also called complementarity determining regions (CDR1 through CDR3), interspersed within four less-variable framework regions (FR1 through FR4) (Fig. 2). V L and V H CDR1-3 are on average 7, 10, and 7 amino acids in size respectively, and more or less centered on AA 33, 55 and 95 respectively. The six CDRs, three each from V L and V H segments, collectively make up the antigen binding site or paratope in a folded Ab molecule. 5

6 Both L and H chains of Ig molecule are folded into distinct domain structures, each of about 110 AAs, called Ig domain (Fig. 3). L chain is folded into two Ig domains, i.e., one variable (V L ) and one constant (C L ), whereas H chain into 4 or 5 Ig domains, i.e., one variable (V H ) and three or four constant (C H 1- C H 3 or C H 4). Each Ig domain contains a loop ( Ig fold ) of about AAs enclosed by intrachain S-S- bonds. Each Ig fold has two intrachain disulphide linked sheets of antiparallel β-strands. In C domains, one sheet of three-strands is exposed to the exterior, while the other of four-strands makes contact with the C domain of the other H or L chain. V domain Ig fold is somewhat different in the sense that the three-stranded sheet (or fivestranded expended sheet) makes contact with the sheet from the other V domain, while the fourstranded sheet lies on the exterior. The three CDRs from V domain of each chain (six CDRs in all) that make up one paratope are located on the outer edges of the five-stranded sheets. The four-stranded sheets of both the C L -C H 1 and C H 3-C H 3 dimers make close contacts, but oligosaccharides covalently bound to C H 2 domains prevent tight packing of C H 2-C H 2. 6

7 Ag-responsive mature B lymphocytes express IgM and IgD as Ag receptor (B cell receptor, BCR) on their surface. But surface IgM is monomeric, unlike pentameric secreted form. Other Igs, i.e, IgG, IgA or IgE expressing B cells are generated during immune response following contact with the Ags. The surface Igs differ from their secreted forms in C-terminal region. The membrane bound Igs have additional spacer, transmembrane and cytoplasmic regions ending in the C-terminus. Surface IgM and IgD have the hydrophobic spacer region of 12 amino acids followed by hydrophobic transmembrane region of 25 amino acids and ending in three hydrophilic amino acids in the cytoplasmic tail (Fig. 4). The surface IgGs have 25 additional amino acids in their cytoplasmic tail, whereas surface IgA has a unique 14 amino acid cytoplasmic tail. The BCR is associated with the signaling components, Ig-α (CD79a) and Ig-β (CD79b) on the B cell membrane for initiating the signal transduction pathways associated with Ag-driven B cell activation. Shapes of antigen-binding site/ paratope Paratope is a three-dimensional shape formed by six CDRs (three each on V L & V H ) brought closer by folding of V L and V H domains together (Fig. 5). Each bivalent Ab has two identical paratopes that can bind two epitopes on multivalent antigens or two haptens. Ig fold of each domain has two layers (interior & exterior) of anti-parallel β-pleated sheets connected by random coils or loops. CDRs lie on the tips of these loops connecting the anti-parallel β-strands. 7

8 Abs produced by different B lymphocyte clones have paratopes of different shapes and complementarity to the antigenic determinants or epitopes so as to achieve the best fit between a paratope and an epitope. Paratope shapes can be as varied as concavities (for accommodating convex or bulging epitopes on protein antigens), long grooves (for epitopes on carbohydrate and nucleic acid antigens), or small pockets (for haptens and monosaccharide epitopes). Polyspecific Abs have however been found that can accommodate more than one type of epitopes within their large polyfunctional binding sites. Dissecting the structure of Abs in order to understand their functions Abs being proteins are amenable to degradation by proteolytic enzymes (proteases). IgG is considered as prototype Ig for studies on structural details. Two proteases, namely papain and pepsin, cleave the human IgG1 to yield fragments of various sizes. Limited digestion with papain cleaves IgG1 in its proximal hinge region to yield: i) two identical N-terminal fragments of about 50 kda size, each being composed of [V L +C L +V H +C H 1] domains, and ii) the C-terminal fragment of about 50 kda size, and composed of [Hinge region + 2(C H 2+ C H 3)]. Each N- terminal fragment is able to bind one hapten molecule or one epitope on a multivalent antigen and therefore designated as Fab (Fragment antigen binding) (Fig. 1). Being monovalent, Fab is not able to agglutinate or precipitate multivalent antigens. These are also not able to bind 8

9 complement component C1q and are therefore unable to activate the complement by classical pathway. On the other hand, C-terminal fragment, being composed of only constant parts of the H chains still joined by disulphide bonds between cysteine residues in the hinge region, can be crystallized, and therefore designated as Fc (Fragment crystallizable). Fc is able to fix C1q, but does not bind antigen. Pepsin degrades human IgG1 into two different fragments: i) one N-terminal large fragment of about 100 kda size, composed of [2(V L +C L +V H +C H 1) + Hinge region], and ii) several small C- terminal peptide fragments, the largest being of about 110 AAs. The N-terminal fragment is functionally bivalent, i.e., can bind two epitopes and cross-link multivalent antigens so as to agglutinate or precipitate them, and therefore designated as F(ab ) 2 (Fig. 1). The largest C- terminal fragment is designated as Fc, just to distinguish it from Fc of the papain digested IgG1. In this manner, these early studies were of immense use for elucidating the structure of Ig molecules and continued efforts culminated into determination of the fine structure of Igs of all major classes from various species. Structure of human and mice Igs is known in more details than those of other species. AA sequencing of myeloma proteins and later of monoclonal antibodies in conjunction with the X-ray crystallographic studies for their three-dimensional conformation, have over past four decades revealed several structural features of Abs that could explain their functions. Nucleotide sequencing of Ig gene segments has allowed faster progress of this field of knowledge. The evolutionarily conserved Ig fold having two layers of β pleated sheets as described in the preceding paragraphs is clearly elucidated in the structures of Ig prototype and other members of a large family of Ig-like proteins, called Ig superfamily (IgSF). A region lying between C H 1 and C H 2 domains of IgG molecules is rich in proline residues that allow rotational and translational flexibility to Fab arms and therefore called hinge region. IgG can adopt Y to T shape due to the mobility in its Fab arms that gives advantage of reaching of the two paratopes to two epitopes of multivalent antigens at varying space. T shape is however more commonly of IgA1 than that of other Igs. Hinge region also has cysteine residues that are involved in disulphide bonding between the two H chains. Hinge region is of variable length in different Ab molecules, even different IgG subclasses, but is lacking in IgM and IgE isotypes. In human IgG1, it has 15 AAs and is divided into proximal, core and distal parts. The core has Cys- Pro-Pro-Cys-Pro sequence. A more elongated Cys- rich hinge region occurs in human IgG3 and account for its functional differences from other IgG subclasses. Human IgA1 has a hinge region of 13 amino acids in length, which is rich in O-linked glycosylation sites and is susceptible to bacterial proteases. Human IgA2 lacks hinge region and is not susceptible to bacterial proteases. A full-sized domain in IgM and IgE each i.e., C H 2 replaces a typical hinge region (these isotypes have four C H domains instead of three in other isotypes). However, overall structures of these Ab molecules have compensations for flexibility. Ab fragments smaller than F(ab ) 2, Fab or Fc, to which some function can be assigned, have now been constructed by the application of recombinant DNA methodologies. One such fragment, composed of [1V H +1V L ] domains, designated as Fv (Fragment variable), harbours one paratope and thus may also be called a single epitope- binder. Still smaller or rather the smallest epitope-binder composed of a single V H domain have recently been constructed and have vast potential for use in therapy, biosensors, proteomics, etc. Such fragments can be expressed in E. 9

10 coli or by phage display system in correctly-folded structure that performs the function of specific antigen binding. Precise information is now available on the binding of different types of antigenic determinants to different paratopes, and also on that of several types of effector molecules (C1q and cell surface Fc receptors) and other ligands to Ab molecules outside their paratopes (Tables 3-5). Fc receptors for IgG, IgA, IgM and IgE have been found to be present on various leukocytes (Table 3). Some proteins of bacterial and viral origin have also been found to act as Fc receptors. Notable among them are: staphylococcal protein A, streptococcal protein G and herpes simplex virus proteins ge and gi (Table 4). In addition, lectins of plant and animal origin, fibronectin, rheumatoid factor (IgM or IgG autoantibody) can bind to Igs in their Fc regions (Table 5). Binding sites of Igs that lie outside the paratope region for several different molecules are precisely known at present. In particular, CH 2 -CH 3 cleft of IgG is promiscuous, i.e., acts as binding site for several different ligands and therefore has control over several functions. Table 3: Various soluble and cell surface receptors which bind in the Fc regions of Igs for effector functions Ig binding effector molecule Present on/ in Binding to I. FcRs in effector functions FcγRIa,b,c (CD64) Most leukocytes Lower hinge-cγ2 FcγRIIa,b (CD32) Most leukocytes Lower hinge-cγ2 FcγRIIIa,b (CD16) Most leukocytes, including NK Lower hinge-cγ2 cells FcαRI (CD89) Monocytes, neutrophils, Cα2- Cα3 interface eosinophils Fcα/µR Mature B lymphocytes &? monocytes FcεRI Basophils, mast cells and some Cε3 other cells FcεRII (CD23) Most leukocytes Cε3 II. Complement binding C1q serum Cµ3 & Cγ2 C3b & C4b (thioester bonds) serum Covalent binding at multiple sites of IgG 10

11 Table 4: Proteins of bacterial and viral origin (microbial FcRs) binding to Igs in their Fc regions Microbial FcR Bacteria/ virus Binding to Ig region Staphylococcal protein A (SpA) Staphylococcus aureus Mammalian Cγ2-Cγ3 interface & also Fab Streptococcal protein G (SpG) Streptococcus pyogenes strains C & G Mammalian Cγ2- Cγ3 cleft & also Fab ge & gi Herpes simplex virus (HSV-1) proteins Human Cγ2- Cγ3 cleft Table 5: Lectins and other ligands binding to Igs in their Fc regions Ligand Source Binding to Ig region Lectins Plants & animals Ig glycans Fibronectin Body fluids & extracellular matrix Fc of aggregated human Igs Rheumatoid factor (IgM/ IgG autoantibodies) Serum & synovial fluid Cγ2- Cγ3 cleft Antibodies vis-a-vis enzymes Basic plan of Ab structure and function is same for all Ab molecules: they are glycoproteins of two identical L chains and two identical H chains or their multimers to bind the antigen specifically and effect its disposal/ destruction. Enzymes are structurally diverse (proteins of different shapes and sizes, ribonucleic acids) but all carry out one basic function of biocatalysis, after binding specifically with their respective substrates. Some Abs have however been found to act as enzymes and are termed as abzymes or catalytic antibodies. Similarities and differences between Abs and enzymes have been summarized in Table 6. Different Ab classes and subclasses are for diverse effector functions Differences in the C L and C H sequences are also responsible for differences in their biological behaviour. Due to differences in C L of L chains, there exist two classes of L chains, designated as κ and λ in nearly all mammalian species. But in any one Ab molecule, only a pair of either κ or λ is found in association with a pair of H chains. The ratio of κ- bearing Igs to λ- bearing Igs varies in different species: κ: λ ratio in mice & rats is 95:5, and in humans 60:40. Differences in C H sequences generate different classes and subclasses of H chains and hence of Abs. In mammals including humans, due to five different H chains, designated as µ, γ, α, ε, and δ, there are respectively five different classes: IgM, IgG, IgA, IgE and IgD. Four subclasses of human IgG, designated as IgG1 (γ1), IgG2 (γ2), IgG3 (γ3) and IgG4 (γ4), and two of IgA, 11

12 designated as IgA1 (α1) and IgA2 (α2) are found in all healthy individuals. In mice, four subclasses of IgG, designated as IgG1 (γ1), IgG2a (γ2a), IgG2b (γ2b) and IgG3 (γ3) are found in all healthy individuals (Table 7). Table 6: Similarities and Differences between Enzymes and Antibodies Feature Enzymes Antibodies Function Biocatalysis Antigen recognition & disposal Chemical Most are proteins, including some Glycoproteins nature Abs (Abzymes); some RNA (ribozymes); Size Wide range (a few kda to MDa) kda (monomers) and 2-6X monomers (multimers) Structure Different for different enzyme (L-H-H-L) 1-6 Binding sites Gain of activity Type of chemical bonds involved Binding Substrate binding site & catalytic site are closely placed to make the active site i) By binding essentially to cofactor/ co-enzyme ii) By conversion of zymogen to active enzyme Non-covalent and covalent bonds with substrate or transition state Ag binding site & effector binding site are distantly placed Fully active in secreted form; Effector binding to C or FcR follows Ag binding Non-covalent bonds with Ag as well as effector molecules Specific to small molecules or small regions of large molecules Specific to small molecules (haptens) or small regions of large molecules Expression Constitutive or induced Induced by contact of B cell Producer cells Different enzymes produced by many different types of cells clone with cognate Ag Ag-specific B cell clones Table 7: Classes and subclasses of immunoglobulins of human and mice Species Immunoglobulin classes & subclasses Mice IgG1, IgG2a, IgG2b, IgA IgM IgE IgD IgG3 Human IgG1, IgG2, IgG3, IgG4 IgA1, IgA2 IgM IgE IgD 12

13 Different classes and subclasses have evolved to perform diverse effector functions of the immune system (Table 8). Abs have basically two distinct regions that perform two distinct functions: a) antigen binding region made up of V L and V H domains, and b) effector binding region, made up of C-terminal 3-4 Ig domains in the H chains that bind effector molecules and cellular receptors. Feature Half-life (days) 7-10 Tissue distribution Complement activation Table 8: Biological properties of human immunoglobulins IgM Mostly in serum Ig class IgGs IgAs (IgA1, IgE IgD (IgG1 to IgG4) IgA2), siga (IgG1, 6 (monomer) 2 3 IgG2, IgG4)? (dimeric siga) 7 days (IgG3) Serum & tissue Serum & mucosal Basophils & Naïve fluids secretions mast cells in mature B tissues cells ++++ IgG3 (+++), IgG1 (++), IgG2 (+), IgG4 ( ) IgG2 & IgG4 mostly (Only alternate pathway by IgA1) Transplacental transfer High affinity Yes - binding to basophils & mast cells Binding to All subclasses microbial but with varying proteins (SpA affinity &SpG) Opsonization Yes, Yes, efficiently Yes efficiently ADCC Yes Virus/ toxin Yes Yes Yes, in mucosal neutralization secretions Bacterial Yes, very Yes Yes, in mucosal agglutination efficiently secretions Structural variants of Abs are genetically determined and antigenically distinguishable Isotypes (Ab classes) and subtypes (Ab subclasses) These are structural variants of L and H chains of Igs, encoded by separate genes present in all healthy individuals of the vertebrate species. For example, mouse has two different isotypes of L chains, designated as κ and λ, and five isotypes of H chains, designated as µ, γ, α, ε, δ. Four 13

14 subtypes of γ exist in mouse and are designated as γ1, γ2a, γ2b, γ3. H chain isotypes and subtypes make different Ig classes (IgM, IgG, IgA, IgE & IgD respectively due to µ, γ, α, ε, δ) and subclasses (IgG1, IgG2a, IgG2b & IgG3 respectively due to γ1, γ2a, γ2b, γ3). Human subclasses of IgG are: IgG1 (γ1), IgG2 (γ2), IgG3 (γ3) and IgG4 (γ4). Human IgA, has two subclasses, namely, IgA1 (α1) and IgA2 (α2). Abs being proteins themselves have all the features of good antigens and induce production of anti-abs when inoculated in suitable individuals other than the self. The isotype- and subtypespecific antigenic determinants on L and H chains are restricted to their respective C regions. Different classes and subclasses of Igs can thus be serologically discriminated by using classspecific and subclass-specific antisera raised in individuals of a different species or specific monoclonal antibodies. Allotypes Structural variants of L and H chain isotypes and subtypes exist within individuals of the same species, because of the presence of alleles of isotype and subtype genes in populations. Allotypes are therefore shared among some but not all individuals of a species e.g., G1m(1) and G1m(3) are human IgG1 allotypes showing sequence differences in C H 1 and C H 3 domains respectively. Most of the allelic differences lie in the constant regions of L and H chain genes, thereby restricting the allotype antigenic determinants to these regions. Allotypes can thus be detected serologically by using anti-allotype allo-antisera (antisera produced by injecting allotypic Ig into the individuals who lack that particular allotype) or specific monoclonal antibody. Idiotypes Structural variants of V regions of L and H chains of Igs that can be detected as antigenic determinants are called idiotypes. Igs produced by different B cell clones in an individual exhibit different idiotypes. So Ig molecules produced by each B clone has its characteristic idiotype. An idiotype is a set of antigenic determinants, called idiotopes, on V regions of L and H chains. Some of the idiotopes are conformational and others are linear. Some of them may also be shared with those on other Igs within the same individual or species or even across the species. Antiidiotype Abs are normally produced in every individual that result in creation of idiotype-antiidiotype networks for regulation of the immune responses. Anti-Id Abs can be produced by immunization of individuals with the Ab of a particular Id or by hybridoma technique. Biological functions of Abs Classical functions of Abs Abs have two major functions i.e., i) antigen recognition by specific binding of Ag in the Agcombining site (paratope), and ii) antigen disposal following Ab binding to various sorts of effector molecules in the regions outside paratope, mostly in their constant domains. Following Ag-Ab complex formation, three major types of effector molecules can bind to Ab leading to Ag disposal by different mechanisms. They are: i) C1q binding to IgM and aggregated IgG molecules leading to activation of the classical pathway of complement, ii) binding to various FcRs on macrophages and other leukocytes for opsonization of the particulate Ags, and also for release of the inflammatory mediators from leukocytes, and iii) binding to FcγRIII on natural killer cells for Ab-dependent cell-mediated cytotoxicity (ADCC). The effector molecules binding 14

15 to Fc regions of different Ig isotypes are presented in table 3. It must however be remembered that Ab-mediated neutralization of toxins and viruses occurs merely by their binding on Ab paratopes. These are examples of Fc region-independent effector function of Abs. In addition, Igs can bind in an Ag- independent manner on the cell surface receptors on epithelial and endothelial cells. Neonatal Fc receptor (FcRn) is responsible for transcytosis of IgG across placenta in humans and rodents, gut epithelium of the colostral IgG in neonates, and in mammary glands secretion of the lactating mammals. FcRn also regulates the levels of IgG in serum. Polymeric Ig receptor (pigr) present on abluminal side of mucosal epithelia allows binding of polymeric Ig isotypes, namely dimeric IgA and pentameric IgM, and transports these molecules on the luminal side. Then it is cleaved to yield a secretory component (SC) that remains associated with the secreted form of the polymeric Ig. SC protects the secretory Ig from proteolytic degradation on the mucosal surfaces. Igs also interact with several different proteins of bacterial and viral origin (Table 5). Protein A of certain strains of Staphylococcus aureus (SpA) and protein G of Streptococcus pyogenes (SpG) are fully characterized for their binding to most subclasses of mammalian IgG in their Fc regions. These so-called microbial FcγRs are structurally different from leukocyte FcγRs, the latter being members of IgSF. SpA also binds in Fab region of mammalian IgGs and acts as a B cell superantigen. Proteins from other bacteria and viruses have also been found to bind on Igs. Microbes use the Ig-binding ability to their advantage, thereby affecting the course of the diseases caused by them. Purification of IgG is often done using SpA- and SpG- affinity chromatography. Lectins of plant and animal origin, and various synthetic ligands have also been found to interact with various parts of Igs outside the paratopes (table 6). Such interactions might have bearing on immunological disorders. Important classical functions of different Ab classes and subclasses IgM IgM is the predominant Ab class in primary immune response and exists mostly as pentamers and some as hexamers. It is very efficient in precipitation, agglutination and opsonization of antigens. It fixes very efficiently complement component C1q after binding with the antigen and triggers C-activation by classical pathway. A single pentameric molecule is able to trigger C activation. Being a large pentameric molecule, it is abundant in serum and negligible in tissue fluids. Its secretion onto mucosal surfaces is mediated by polymeric Ig receptor (pigr). IgG IgG is the predominant Ab class in late primary and secondary (booster) humoral immune responses and remains in circulation for longer time than any other Ab class. It is efficient in precipitation, agglutination and opsonization, but less effective than the pentameric decavalent IgM. It fixes complement component C1q after binding with the antigen and triggers classical pathway of C-activation. But aggregation of IgG molecules on the antigen is required for C1q binding. Its concentration is the highest of all isotypes. It occurs in almost equal amounts in both serum and tissue fluids. Unlike humans and rodents, it is secreted in colostrum and milk of ruminants in more amount than that of IgA. Different subclasses of IgG show variation in their effector functions (Table 8). 15

16 IgA IgA is abundant in mucosal secretions and the primary mediator of mucosal immunity. Colostrum is rich in IgA. It is present in more amounts than other Ab classes in milk in rodents and humans. It is mainly synthesized in gut and respiratory tract mucosa-associated lymphoid tissues (MALT). In the mucosal secretions, it mainly exists as a dimer joined by a J chain and held together by a secretory component (SC), and designated as SIgA (secretory IgA) (Fig. 6). Human IgA1 subclass is more abundant than IgA2 in serum and secretions, except in colon where IgA2 has more concentration. SIgA protect mucosal surfaces from pathogens by excluding their entry, agglutinating them, interfering with flagellar motility and neutralizing microbial toxins at these sites. Systemic IgA mediates opsonization of microbial Ags by engagement of FcαRI on phagocytes. It also induces respiratory burst activity, and release of cytokines and proinflammatory mediators from phagocytes. IgE This antibody class is involved in allergies (type I hypersensitivity) and parasitic immunity. Unlike other Ab classes, it is heat-labile and inactivated at 56ºC in 30 min. It binds via high affinity FcεRI on basophils and mast cells in tissues, where it remains in bound form, and is therefore also called cytophilic antibody. Allergen binding by these cytophilic IgE Abs on mast cells causes release of the granular contents that are the inflammatory mediators of type I hypersensitivity. It is also called reaginic antibody for its involvement in allergies. It binds to other leukocytes via low affinity FcεRII for its role in immune response generation. 16

17 IgD IgD is cell surface-associated BCR, in addition to IgM monomer on mature B lymphocytes, and is of the same specificity for Ag as that of IgM. Its role in immunity is not clearly understood, but is possibly involved in increasing the efficiency of B cell activation following Ag binding onto the B cell. Non-classical functions of Abs Recently, several V-domain mediated functions of Abs, in addition to Ag binding, have been found. These are: catalysis, nucleotide binding, self-binding and superantigen binding. The conserved segments of the V domains mediate most of these functions, but their biological significance is not clearly understood at present. Different subsets of Abs show these nonclassical functions and a term superantibody has been proposed in order to distinguish them from the conventional Ag-binding function of Abs. Superantibody activities may be involved in autoimmune disease and protection against infections. For example, levels of certain catalytic Abs are increased in autoimmune diseases. These novel activities may also find applications in biotechnology and medicine. Box 2: In vivo biological roles of antibodies Ab neutralizes viruses and toxins by binding with them specifically and blocking virus entry in the cells or toxin binding on cellular receptors Abs complexed with Ags activate classical pathway of complement leading to lysis of the membrane-bound Ags such as bacteria, enveloped viruses and other pathogens Abs act as opsonins to enhance phagocytosis of the Ab-coated Ags Abs link cell surface Ags to NK cells for Ab-dependent cellular cytotoxicity Abs can act as positive and negative regulators of inflammation and cell-mediated immunity Abs are involved in mediating hypersensitivities (IgE in type I, IgG in type II and type III reactions) Abs are involved in autoimmunity Abs are involved in removal of effete cells & debris during normal wear and tear in the body Abs exert direct anti-microbial effects by binding on surface proteins and causing interference with microbial physiology, thereby acting somewhat as antibiotics Abs can act as positive and negative regulators of inflammation and cell-mediated immunity. Complement (C ) activation by Ag-Ab complexes produces proinflammatory C components. Cross-linking of FcR by immune complexes promotes phagocytosis by macrophages. Specific Abs can influence inflammatory response by removal of microbial antigens that have proinflammatory or anti-inflammatory effect in the host. IgM are often proinflammatory due to their pronounced C activating ability. IgGs can either be pro- or anti-inflammatory, depending on their subclass, concentration, and interactions with stimulatory or inhibitory FcRs. Abs have also been shown to exert direct antimicrobial effects. By binding on surface proteins, Abs cause interference with microbial physiology and exert anti-microbial effects. In this manner, they act somewhat as antibiotics. For example, anti- E. coli lipopolysaccharide (LPS) 17

18 antibodies interfere with enterochelin release, thereby preventing iron acquisition by bacteria leading to bacteriostatic effect. Abs, when given as Ab-Ag complexes, regulate the immune response to the Ag. IgM-Ag complexes often augment the immune response against the antigen. These immune complexes activate C resulting in binding of C3b component on the Ag. This complex would then be trapped by complement receptors (CRs) on the surface of follicular dendritic cells (FDCs) in the germinal centers. Thus specific B cell clone coming to the site makes contact with the Ag trapped on the FDCs. IgG-Ag complexes are often found to have inhibitory effect on immune response development. It could be that IgG acts as blocking Ab (Ag masking Ab) or inhibits immune response in an FcR-mediated inhibition of B cell activation. Abs as biocatalysts An antibody that can act as a catalyst for a chemical reaction, in addition to its specific binding to the antigen, is termed as catalytic antibody or abzyme. More than 100 different monoclonal antibodies having catalytic activity have been reported that carry out as diverse reactions as pericyclic processes, elimination reactions, hydrolyses, bond-forming reactions and redox reactions. Bence-Jones proteins from multiple myelomas and L chains of certain autoabs can express peptidase activities. VIPases, DNases, esterases and thyroglobulinase activities have been described in abzymes. Haptens and peptide Ags can induce catalytic Ab synthesis. The transition state analogues can induce the production of antibodies. These Abs in their active sites bind to the transition state more strongly than the substrate. Further genetic manipulation introduces catalytic residues in their active sites. Like enzymes, Abzymes process their substrates through a Michaelis complex in which the chemical transformation occurs followed by product dissociation. The steady-state kinetics for all abzymes obey the Michaelis-Menten rate expression. The value of k cat / k uncat for abzymes is generally in the range of 10 1 to It is very difficult to demonstrate catalytic activity of antibodies in antisera, because of their heterogeneity. Monoclonal antibodies and recently phage display library antibody fragments are therefore used for searching catalytic activities in them. Cat ELISA is a test used for screening of abzymes, in which the product formed during catalysis is captured by another product-specific antibody that is then detected by ELISA amplification system. Abzymes are still in infancy, but are promising in the sense that they can carry out certain chemical reactions more efficiently than the classical enzymes do. Forces involved in antibody-antigen interactions Antibody binds to the antigen specifically in its paratope, involving various types of noncovalent bonds. They are listed below: 1. Ionic bonds or electrostatic interactions typically between COO and NH 3 + groups on ionized amino acids of interacting paratope and epitope. These bonds are most influenced by ph and ionic strength of the medium. Bonding energy is in the range of 5-8 kcals/ mole. 2. Hydrogen bonds between O H and NH 2 groups of interacting paratope and epitope. Bonding energy is 1-2 kcals/ mole. 18

19 3. van der Waal s forces, very weak forces due to electron clouds of atoms and molecules that have come very close to each other. Bonding energy is ~1 kcal/ mole. 4. Hydrophobic interactions due to exclusion of water or polar solvent molecules from the two non-polar moieties approaching each other. Bonding energy is ~5 kcals/ mole. About 50% of the binding strength comes from these interactions. Box 3. Forces involved in Ag-Ab interaction Only non-covalent reversible bonds are formed between Ags & Abs, which are: Ionic interactions (5-8 kcal/ mole) Hydrophobic interactions (5 kcal/ mole) Hydrogen bonds (1-2 kcal/ mole) van der Waals forces (1 kcal/ mole) Although covalent bonds are not involved in Ab-Ag interactions, multiple non-covalent bonds have good amount of bonding energy for providing overall stability to Ab-Ag complexes. Unlike covalent bonds, non-covalent binding of paratope and epitope is reversible and the net outcome of difference between total attractive and total repulsive forces. Therefore, Ab-Ag interactions are explained by using the law of mass action. Ab-Ag interactions are usually exothermic at room temperature or lower temperatures. Obligatory long-range attraction between paratope and epitope also play their role in Ab-Ag interactions. Biopolymers repel each other and no two molecules can come nearer than 3-5 nm to each other. Paratope and epitope can however penetrate the repulsion fields of each other and come within 3 nm of each other due to their shape complementarities, opposite electrostatic charges and hydrophobicity of the paratope. Affinity, avidity and specificity of antibodies Antibody affinity Affinity is a thermodynamic parameter to quantify the strength of binding of two interacting molecules in solution. Antibody affinity is thus the strength of binding of a single paratope with a single epitope in appropriate solution. Ab affinity is also called intrinsic affinity and depends on structural complementarity of paratope and epitope. Small changes in the structure of paratope affect the Ab affinity. Affinities of different Abs range between 10 5 / M (low affinity) to / M (high affinity). Ab affinity matures or improves in the developing immune response against the inducing antigen by the process of somatic mutations in CDRs of the Ig V gene segments in B lymphoblasts. B lymphocyte dividing by mitosis following antigen recognition is called B lymphoblast. Ab affinity can be measured experimentally (by equilibrium dialysis, radio-immunoassay, competitive ELISA, etc.), wherein a single species of Ab molecules and a monovalent epitope or hapten are used. According to the law of mass action, K A = [Ab.Ag]/ [Ab][Ag] Where, K A is the affinity constant, [Ab.Ag] is the conc. of Ab-Ag complex, 19

20 [Ab] is the conc. of free Ab, and [Ag] is the conc. of free Ag Ab affinity is measured using Scatchard equation or other suitable equations. Scatchard equation: K= r / (n-r) (c) Where, r = Conc. of bound Ag (ligand)/ Conc. of total Ab molecules in the system n = Ab valency (= 2 for IgG), c = Conc. of free or unbound ligand. Monoclonal antibodies are homogeneous i.e. each and every monoclonal Ab molecule in a solution is of the same affinity for the antigen. Whereas, Abs against a particular epitope present in antisera are heterogeneous i.e., Abs produced by the B lymphocyte clones in a developing immune response have slight variations in their affinities against the same epitope. Such variations in Ab affinities are introduced by somatic mutations in CDRs of Ig V gene segments in the mitotically dividing B lymphoblasts. Practical importance of Ab affinity In vivo reactions carried out by high affinity Abs are functionally better than those by low affinity Abs, since latter are difficult to be removed from the body and may lead to pathological conditions. High affinity Abs are more useful in serological tests in vitro as they ensure more specificity. Moderate affinity Abs are however more useful in Ab affinity- based purification techniques. Antibody avidity/ functional affinity Ab avidity is the total strength of binding of a multivalent Ab with a multivalent antigen. Ab avidity is governed by the intrinsic affinity of Ab, valencies of Ab and Ag, and stereic hindrance of the interacting molecules. Ab avidity is of more practical consequences than intrinsic affinity. Antibody specificity and cross-reactivity Ab specificity is the ability of Ab molecules to discriminate between the binding of the inducing Ag and that of the antigens chemically related to the inducing one. Ab specificity is governed by the structural (geometrical shape) and chemical (bonding) complementarities between the Ab paratope and the Ag determinant. The binding of Ab to the inducing Ag generally represents the best fit between Ab paratope and the epitope on the inducing Ag. In other words, the Ag that induces the production of specific Ab binds to this Ab with the highest affinity in the tightest possible manner. The epitope on the inducing Ag is called the homologous Ag determinant and the determinants chemically related to the homologous one are called the heterologous or cross-reactive determinants. Some exceptions to this rule exist in that certain heterlogous Ags can bind with higher affinity to an Ab induced by homologous Ag. Such Abs are called heteroclitic Abs. Ag determinants that are chemically different but bind with the homologous Ab are known as mimotopes e.g. peptides mimicking the carbohydrate epitopes for binding to the anti-carbohydrate Ab. Abs display exquisite specificity for the antigens i.e., they can discriminate between stereoisomers, optical isomers and charge differences due to variations in the primary structure of the closely related Ags. 20

21 Box 4: Ab affinity, avidity and specificity Antibody affinity is the strength of binding of a single paratope with a single epitope Ab affinities range between 10 5 / M (low) and / M (high) Ab affinity is measured by using Scatchard equation: K= r/ (n-r).c Ab avidity is the total strength of binding of a multivalent Ab with a multivalent antigen Ab specificity is the ability of Ab molecules to discriminate between the binding of the inducing Ag and that of the antigens chemically related to the inducing one Antisera represent complex mixtures of antibody molecules of polyclonal origins Chemically related epitopes and antigens can bind to homologous Ab, but with lower affinities than that of homologous Ag:Ab pair Epitopic cross-reactivity with homologous Ab results from reduced structural and chemical complementarity of heterologous epitopes for homologous Ab paratope Cross-reactivity of multivalent antigens (antigenic cross-reactivity) with homologous antisera may result from both epitopic cross-reactivity and epitope sharing between homologous and heterologous Ags Antisera represent complex mixtures of antibody molecules of polyclonal origins. They show many different specificities which arise in two different ways: i) Two or more different Ags given simultaneously in the same animal will result in production of Abs against all of them and therefore the antiserum would react against all of them, ii) Most Ags existing in Nature are multivalent and therefore different Abs are produced against different epitopes of the multivalent Ag. These Abs originate from different B cell clones and therefore antisera contain a mixture of polyclonal Abs. The cross-reactive Ags share some of the epitopes with the homologous Ags are therefore able to bind with Ag-specific Ab in the antisera. Chemically related epitopes and antigens can thus bind to homologous Ab, but with lower affinities than that of homologous Ag-Ab pair. In other words, we can say that epitopic crossreactivity with homologous Ab results from reduced structural and chemical complementarity of heterologous epitopes for homologous Ab paratope. On the other hand, cross-reactivity of multivalent antigens (antigenic cross-reactivity) with homologous antisera containing homologous Abs against different epitopes on the homologous Ag may result from both epitopic cross-reactivity and epitope sharing between homologous and heterologous Ags. Ig genes and generation of antibody diversity The extent of antibody diversity No two immunoglobulin molecules produced by two different B lymphocyte clones are alike. A huge number of different B lymphocyte clones exist in healthy individuals of all vertebrate species that bear immune system. The clones are different for they bear different antigen-specific B cell receptors (BCRs) on their surface. The number of B cell clones in different species is 21

22 however different, depending on the size of the species and blood composition. For example, an adult mouse has about 10 7 B cells and therefore, a possible maximum of 10 7 B cell clones producing 10 7 different Abs that bind specifically with an equal number of different Ags. However, the number of antigens in the whole universe is enormous, and therefore 10 7 different Abs make a relatively limited number for the species to cope up with the vastness of the antigenic world. Nevertheless, molecular biology studies during past three decades have clearly suggested that using genetic information contained in segmented genes for L and H chains, molecular mechanisms can potentially produce diverse Abs of the order of Then, how can a mouse produce antibody against any imaginable Ag introduced into it? One possible answer lies in the fact that BCR can bind specifically with one Ag and the same can also bind with cross-reactive Ags initially giving sufficient threshold signal to trigger the immune response. Subsequent to this initial event, the developing immune response improves Ab affinity against the inducing Ag by somatic hypermutation. This may bring the figure to 10 9 different Abs at the most. Recently discovered polyspecific antibodies may further solve this puzzle to some extent. But we also remember at the same time that any individual encounters only a limited number of Ags in its surroundings during its lifetime and this limit can be of the order of millions. Which antigens will be encountered at what time in an individual is however not predetermined to a large extent and the individuals are not pre-cognizant of the Ags in the changing environment! Ig genes and their genomic organization Ig is composed of two identical IgH chains and two identical IgL chains derived from Igκ or Igλ gene segments. The murine IgH locus has upto a thousand V gene segments in about 1 Mb region that begins about 100 kb upstream of Cµ on chromosome 12. A cluster of 4 J gene segments lie about 7.5 kb upstream of Cµ. Between V H and J H are present 13 D gene segments. V H gene segments at their 3 - ends and J H gene segments at their 5 -ends are flanked by recombination signal sequences (RSS) containing 23-bp spacer sequence. D gene segments are flanked on both sides by RSS with 12-bp spacer sequence. Eight C H genes are located downstream of the VDJ gene segments in a 200 kb region on chromosome 12. The order of murine C H genes starting downstream of J H is 5 Cµ-Cδ-Cγ3-Cγ1-Cγ2b-Cγ2a-Cε-Cα- 3. As Cµ is the first to recombine with a functionally rearranged V H (D)J H exon, IgM is the first isotype expressed on the B cell surface. Alternative RNA splicing of the V(D)J H exon to the C exons leads to the expression of IgD. Thus, both IgM and IgD are expressed on naïve mature B cells. All other isotypes are produced by class switch mechanism described elsewhere. Switch regions are present upstream of every C gene except Cδ. Each C region gene has a separate exon for each C H domain and hinge region and an additional M exon for cell membrane-bound form of Igs. Igκ locus is about 3 Mb size on murine chromosome 6 and has 140 Vκ, and 4 functional and one non-functional Jκ gene segments just upstream of a single Cκ gene. Unlike V H and Igλ, Vκ gene segments are in both transcriptional orientations and are rearranged by both deletions and inversion of intervening sequences. Vκ gene segments are flanked by 12-bp RSS and Jκ by 23-bp RSS. 22

23 Igλ locus is about 200 kb on murine chromosome 16 and has 3 functional Vλ flanking 23-bp RSS. Upstream of 3 functional and one non-functional Cλ are Jλ gene segments flanked by 12- bp RSS. Genomic organization of mouse IgH, Igκ and Igλ loci are shown in Figure 7. 23