Immunology Notes PDF

SECTION Immunology 2 https://t.me/docinmayking SECTION OUTLINE 9. Immunity (Innate and Acquired) 10. Antige...

SECTION Immunology 2 https://t.me/docinmayking SECTION OUTLINE 9. Immunity (Innate and Acquired) 10. Antigen 11. Antibody 12. Antigen–antibody Reaction 13. Complement 14. Components of Immune System: Organs, Cells and Products 15. Immune Responses: Cell-mediated and Antibody-mediated 16. Hypersensitivity 17. Autoimmunity 18. Immunodeficiency Disorders 19. Transplant and Cancer Immunology 20. Immunoprophylaxis https://t.me/docinmayking  CHAPTER Immunity (Innate and Acquired) 9 CHAPTER PREVIEW „„Innate Immunity „„Bridges Between Innate and Acquired „Herd Immunity „„Acquired or Adaptive Immunity Immunity „Adoptive Immunity „Active Immunity „„Other Types of Immunity „Passive Immunity „Local (or Mucosal) Immunity The term “immunity” (Latin word “immunitas”, meaning of infection (Table 9.1). The first step that takes place freedom from disease) is defined as the resistance offered is attachment, which involves binding of the surface https://t.me/docinmayking by the host against microorganism(s) or any foreign molecules of microorganisms to the receptors on the cells substance(s). Immunity can be broadly classified into two of innate immunity. types: 1. Innate immunity—present right from birth Microbial Surface Molecules 2. Acquired/adaptive immunity—acquired during the They are the repeating patterns of conserved molecules course of the life. which are common to most microbial surfaces; called Microbes-associated molecular patterns (MAMPs). Examples of MAMPs include peptidoglycan, INNATE IMMUNITY lipopolysaccharides (LPS), teichoic acid and lipoproteins Innate immunity is the inborn resistance against infections present on bacterial surface. that an individual possesses right from the birth, due to his genetic or constitutional makeup. Pattern Recognition Receptors (PRRs) Innate immunity has certain unique properties by which These are the molecules present on the surface of host cells it can be differentiated from acquired immunity (Table 9.1). (e.g. phagocytes) that recognize MAMPs. They are generally ™ Acts in minutes: Innate immunity is the first line of host conserved regions, encoded by germ line genes. defense against infections; occurs immediately after the ™ Toll-like receptors (TLRs) are classical examples of microbial entry pattern recognition receptors, named after the fruit fly ™ Prior microbial exposure is not required: Innate (Drosophila); the main receptor for inducing innate immunity is independent of prior exposure to the microbes; immunity present even before the first entry of the microorganism ™ Signals generated following binding of TLRs to MAMPs ™ Diversity is limited: Innate immunity is active only activate transcription factors that stimulate expression against a limited repertoire of antigens; in contrast to of genes encoding cytokines and enzymes, which are acquired immunity which is more varied and involves involved in several antimicrobial activities of cells of specialized immune responses innate immunity. ™ Non-specific: Cells of innate immunity are non-specific in their action; can be directed against any microbial Components of Innate Immunity antigen(s) There are several mediators of innate immunity. They ™ No memory: Innate immunity does not have a memory exert antimicrobial activities by various mechanisms as component. Response to a repeat infection is identical described below. Some of these mediators are not purely to the primary response. part of innate immunity; they often act as bridge between innate and acquired immunity (e.g. complements and MECHANISMS OF INNATE IMMUNITY macrophages). Receptor Interaction Anatomical and Physiological Barriers Following the exposure to microorganisms, several ™ Anatomical barriers such as skin and mucosal surfaces mediators of innate immunity are recruited to the site have a spectrum of antimicrobial activities (Table 9.2) 134 SECTION 2  Immunology Table 9.1: Differences between innate and acquired immunity. Innate immunity Acquired/Adaptive immunity Resistance to infection that an individual possesses from birth Resistance to infection that an individual acquires during his lifetime Immune response occurs in minutes Immune response occurs in days Prior exposure to the antigen is not required Develops following the antigenic exposure Diversity is limited, acts through a restricted set of reactions More varied and specialized responses Immunological memory responses are absent Immunological memory responses are present Microbial antigen: Innate immunity develops against antigens that are Microbial antigen: Acquired immunity develops against shared by many microbes (called microbes-associated molecular patterns) antigens that are specific for each microbes Host cell receptors of innate immunity (called pattern recognition Host cell receptors are specific, e.g. T cell receptors and B cell receptors) are non-specific, e.g. Toll-like receptor immunoglobulin receptors Components of innate immunity Components of acquired immunity Anatomical barriers such as skin and mucosa T cell Physiological barriers (e.g. body temperature) B cell Phagocytes (neutrophils, macrophages and monocytes) Classical complement pathway Natural killer (NK) cells Cytokines (IL-2, IL-4, IL-5, IFN- ) Other classes of lymphocytes: T cells, NK-T cells B-1 cells and marginal-zone B cells Types of acquired immunity https://t.me/docinmayking Mast cells, macrophages and dendritic cells It can be classified in two ways: Complement pathways—alternative and mannose binding pathways Active and passive immunity Fever and inflammatory responses Artificial and natural immunity Normal resident flora Cytokines: TNF- , interleukins (IL-1, IL-6, IL-8, IL-12, IL-16, IL-18), IFN- , and TGF- Acute phase reactant proteins (APRs) Abbreviations: TNF, tumor necrosis factor; TGF-β, transforming growth factor-β; IFN, interferon; IL, interleukin. ™ Physiological barriers that contribute to the innate ™ NK-T cells: They are present in epithelium and lymphoid immunity are the body temperature, pH and various organs soluble secretory products of mucosa (Table 9.2). ™ B-1 cells: They are found mostly in the peritoneal cavity and mucosal tissues Phagocytes ™ Marginal-zone B cells: They are present at the edges of Phagocytes such as neutrophils, macrophages lymphoid follicles of spleen. including monocytes are the main components of innate immunity. They are rapidly recruited to the site of Mast Cells infection. Phagocytosis involves three sequential steps— They are present in the epithelial lining of the respiratory (1) engulfment of microbes and subsequent hosting in and other mucosa. phagosome, (2) fusion of lysosome with phagosome to ™ They are activated by microbial products binding to toll- form phagolysosome and (3) microbial killing (described like receptors or by IgE antibody dependent mechanism, in Chapter 14). following which; ™ They release abundant cytoplasmic granules rich in Natural Killer (NK) Cells histamine, prostaglandins and cytokines that initiate They are a class of lymphocytes that kill virus infected cells inflammation and proteolytic enzymes that results in and tumor cells. NK cell mediated mechanism of killing killing of bacteria. microbes is described in Chapter 15. Dendritic Cells Other Rare Classes of Lymphocytes They respond to microbes by producing numerous T and B lymphocytes are the chief mediators of acquired cytokines that initiate inflammation. They also serve immunity. However, there are several rare types of as vehicle in transporting the antigen(s) from the lymphocytes that share the features of both acquired and skin and mucosal sites to lymph nodes where they innate immunity (Described in detail in Chapter 14), e.g. present the antigen(s) to T cells. Hence, dendritic ™ T cells (also called intraepithelial lymphocytes): They cells serve as a bridge between innate and acquired are present in epithelial lining of skin and mucosa immunity.  CHAPTER 9  Immunity (Innate and Acquired) 135 Table 9.2: Role of barriers in innate immunity. Anatomical Function barrier Skin barrier Mechanically prevents entry of microbes Produces sebum containing antimicrobial peptides and fatty acids Killing of microbes by intraepithelial lymphocytes Mucosal barrier Mucous Prevents entry of microbes mechanically and by membrane producing mucus which entraps microbes Cilia Cilia present in the lower respiratory tract propel the microbes outside Normal flora Intestinal and respiratory mucosa are lined by normal flora Physiological Function barrier Temperature Normal body temperature inhibits the growth of some microbes Fig. 9.1: Major events in an inflammatory response. https://t.me/docinmayking Low pH Gastric acidity inhibits most of the microbes or other foreign substances. The major events that take Secretory products of mucosa place during an inflammatory response following a Saliva Enzymes in saliva damage the cell wall and cell microbial entry are as follows (Fig. 9.1): membrane of bacteria ™ Vasodilation due to release of vasoactive substances Tears Contains lysozyme that destroys the from the damaged tissues peptidoglycan layer in bacterial cell wall ™ Leakage of plasma proteins through blood vessels Gastric juice HCl kills microbes by its low pH ™ Recruitment of phagocytes (e.g. neutrophils) to the site Trypsin Hydrolyses bacterial protein of inflammation—phagocytes undergo the following Bile salts Interfere with bacterial cell membrane steps—(1) margination (adherence to the endothelium), Fatty acids Denature the bacterial proteins (2) rolling on endothelium, (3) extravasation (moves out Spermine Present in semen, inhibits growth of of the blood vessels), (4) chemotactic migration to the gram-positive bacteria inflammation site Lactoferrin Binds to iron, thus interferes with acquisition of ™ Engulfment of microbes and dead material by the iron by bacteria phagocytes ™ Destruction of the microbes. Complement Pathways Inflammation is not always protective in nature, Alternative and mannose binding pathways are the chief sometime, it may produce injurious consequences to host mediators of innate immunity. tissues in the form of hypersensitivity reactions. ™ Alternative complement pathway is activated in response to bacterial endotoxin whereas the mannose binding Normal Resident Flora pathway is stimulated by mannose carbohydrate residues Normal resident flora lining intestinal, respiratory on bacterial surface. and genital tract exert several antimicrobial activities. ™ Following activation, the complements mediate various (described in Chapter 7) biological functions such as (refer Chapter 13): ™ They compete with the pathogens for nutrition „ Lysis of the target microbes (by forming pores on the ™ They produce antibacterial substances. microbial surfaces) „ Stimulate inflammation (by secreting inflammatory Cytokines mediators) In response to the microbial antigens, dendritic cells, „ Stimulate acquired immunity: Complements macrophages, and other cells secrete several cytokines that are another bridge between innate and acquired mediate many of the cellular reactions of innate immunity immunity. such as: ™ Tumor necrosis factor- (TNF- ) Inflammatory Response ™ Interleukin-1 (IL-1), IL-6, IL-8, IL-12 and IL-16 Inflammation is defined as the biological response of ™ Interferons (IFN- , ) and vascular tissues to harmful stimuli, such as microorganisms ™ Transforming growth factor (TGF- ). 136 SECTION 2  Immunology Acute Phase Reactant Proteins (APRs) Contd... They are the proteins synthesized by liver at steady ‰ Marked increase of CRP (>10 mg/dL): It occurs in concentration, but their synthesis either increases or conditions such as acute bacterial infections, major trauma decreases exponentially during acute inflammatory and systemic vasculitis. conditions. Though liver is the primary site, APRs can also CRP Can be Detected by be synthesized by various other cells such as endothelial ‰ Precipitation method using C-carbohydrate antigen cells, fibroblasts, monocytes and adipocytes. (obsolete, not in use now) ™ Positive APRs: They are the proteins whose levels ‰ Latex (passive) agglutination test using latex particles increase during acute inflammation. Examples include: coated with anti-CRP antibodies „ Serum amyloid A It is the most widely used method employed worldwide Detection limit of CRP by latex agglutination test is „ C-Reactive protein 0.6 mg/dL. „ Complement proteins—complement factors (C1– Highly Sensitive CRP (hs-CRP) Test C9), factor B, D, and properdin Minute quantities of CRP can be detected by various methods „ Coagulation protein, e.g. fibrinogen, von Willebrand (e.g. nephelometry, enzyme immunoassays). This is useful in factor assessing the risk to cardiovascular diseases. „ Proteinase inhibitors, e.g. -1 antitrypsin „ 1 acid glycoprotein „ Mannose binding protein ACQUIRED OR ADAPTIVE IMMUNITY „ Haptoglobin Acquired immunity is defined as the resistance against the „ Metal binding proteins, e.g. ceruloplasmin. https://t.me/docinmayking infecting foreign substance that an individual acquires or ™ Negative APRs: They are the proteins whose levels are adapts during the course of his life. decreased during acute inflammation; thus creating a Acquired immunity has unique properties by which it negative feedback that stimulates the liver to produce can be differentiated from innate immunity (see Table 9.1). positive APRs. Examples of negative APRs include ™ Mediators: T cells and B cells are the chief mediators of albumin, transferrin and antithrombin acquired immunity. Other mediators include: ™ Role of APRs: They have a wide range of activities that „ Classical complement pathway contribute to the host defense „ Antigen presenting cells „ APRs have various antimicrobial and anti- „ Cytokines (IL-2, IL-4, IL-5). inflammatory activities (e.g. complement factors) ™ Response occurs in days: Acquired immunity involves „ Metal binding proteins can chelate various metals activation of T and B cells against the microbial antigens; such as iron, copper, etc. making them unavailable which takes several days to weeks to develop, following for the bacteria. the microbial entry ™ Requires prior microbial exposure: Acquired immunity C-Reactive Protein (CRP) develops only after the exposure to the microbes. It is not C-reactive protein is an example of APR that rise in acute inflammatory conditions including bacterial infections. It present prior to the first contact with the microbes belongs to beta globulin family. ™ Specific: Acquired immunity is highly specific; directed ‰ CRP is so named because it precipitates with C- carbohydrate against specific antigens that are unique to the microbes (polysaccharide) antigen of pneumococcus. However, ™ Memory present: Acquired immunity does have a it is not an antibody against the C-carbohydrate antigen memory component. A proportion of T and B cells of pneumococcus; it is non-specific, can be raised in any become memory cells following primary contact of the inflammatory conditions microbe, which play an important role when the microbe ‰ It is one of the most common markers of acute inflammation, is encountered subsequently used in most diagnostic laboratories. ™ Diversity is wide: Acquired immunity though takes time ‰ Following an injury or inflammation, the CRP level starts increasing by 6 hr, doubles every 8 hr, reaches its peak by to develop, is active against a wide range of repertoire of 48 hr. antigens ™ Host cell receptors of acquired immunity are specific CRP Level The normal level of CRP is less than 0.2 mg/dL. However, it for a particular microbial antigen increases by several folds in acute inflammatory conditions. „ Examples include T cell receptors and B cell ‰ Insignificant increase of CRP (10,000 Dalton oral administration of antigens molecular weight, e.g. hemoglobin); more potent is the „ Inhalation of pollen antigens induces IgE synthesis; molecule as an immunogen whereas the same antigens given parenterally, elicits ™ Chemical nature of the antigen: Proteins are stronger IgG antibodies immunogens than carbohydrates followed by lipids and „ Site of injection may influence immunogenicity: The nucleic acids hepatitis B vaccine is more immunogenic following ™ Susceptibility of antigen to tissue enzymes: Only deltoid injection than gluteal injection. This may be substances that are susceptible to the action of tissue due to the paucity of antigen presenting cells (APCs) enzymes are immunogenic. Degradation of the antigen in gluteal fat. by the tissue enzymes produces several immunogenic ™ Repeated doses of antigens: Sometimes to generate an fragments having more number of epitopes exposed. adequate immune response, repeated doses of antigens Molecules that are not susceptible to tissue enzymes over a period of time may be needed. However, after such as polystyrene latex composed of D-amino acids certain doses of antigens, no further increase in antibody are not antigenic; while polypeptides consisting of response is seen L-amino acids are antigenic as they are degradable by ™ Multiple antigens: When two or more antigens are tissue enzymes administered simultaneously, the effects may vary. The ™ Structural complexity: Polymers made up of atleast antibody response to one or the other antigen may be two or more amino acids are immunogenic. Complex equal or diminished (due to antigenic competition) 142 SECTION 2  Immunology or enhanced (due to adjuvant like action, see below Table 10.1: Differences between T-independent antigens and highlight box) T-dependent antigen. ™ Effect of prior administration of antibody: The T-independent antigen T-dependent antigen immune response against a particular antigen Structurally simple—LPS, capsular Structurally complex— is suppressed if its corresponding antibody was polysaccharide, flagella protein in nature administered prior to that Dose dependent immunogenicity Immunogenic over wide „ The primary immune response is more susceptible to range of doses get suppressed than the secondary immune response No memory Memory present „ Therapeutic application: In Rh negative women No antigen processing Antigen processing step is carrying an Rh positive fetus, the anti-Rh globulin is needed administrated immediately following delivery (within Slowly metabolized Rapidly metabolized 72 hours) which prevents the Rh sensitization in Rh Activate B cells polyclonally Activate B cells monoclonally negative women by a negative feedback mechanism. Activate both mature and Activate mature B cells only immature B cells Adjuvant B cells stimulated against T-inde- B cells stimulated against T- The term “adjuvant” refers to any substance that enhances pendent antigen do not undergo dependent antigen undergo the immunogenicity of an antigen. They are usually added to Affinity maturation Affinity maturation vaccines to increase the immunogenicity of the vaccine antigen. Class switch over Class switch over Examples of Adjuvant Activity Antibody response is restricted to Antibodies of all classes can ‰ Alum (aluminum hydroxide or phosphate) IgM and IgG3 be produced https://t.me/docinmayking ‰ Mineral oil (liquid paraffin) Abbreviations: LPS, lipopolysaccharides; Ig, immunoglobulin. ‰ Freund’s incomplete adjuvant: It is a water-in-oil emulsion containing a protein antigen in the aqueous phase ‰ Freund’s complete adjuvant is the mixture of Freund’s T-dependent (TD) Antigens incomplete adjuvant and suspension of killed tubercle Most of the normal antigens are T cell dependent, they bacilli in the oil phase are processed and presented by antigen-presenting cells ‰ Lipopolysaccharide (LPS) fraction of gram-negative bacilli, (APCs) to T cells which leads to T cell activation. The e.g. LPS of Bordetella pertussis acts as an excellent adjuvant activated T cells secrete cytokines that in turn stimulate for diphtheria and tetanus toxoids. This explains the reason for using combined immunization for diphtheria, pertussis the B cells to produce antibodies. and tetanus in the form of DPT vaccine ‰ Other bacteria or their products: T-independent (TI) Antigens Mycobacterium bovis There are a few antigens such as bacterial capsule, flagella Toxoid (diphtheria toxoid and tetanus toxoid act as and LPS (lipopolysaccharide) that do not need the help of adjuvant for Haemophilus influenzae—type b vaccine). T cells and APCs. They directly bind to immunoglobulin ‰ Nonbacterial products: Such as silica particles, beryllium receptors present on B cells and stimulate B cells sulfate, squalene and thiomersal. polyclonally. It leads to increased secretion of non-specific Mechanism of Adjuvant Action antibodies (i.e. hypergammaglobulinemia). Adjuvants act through the following steps: Detailed mechanism of B cell activation against TD ‰ Delaying the release of antigen: Adjuvant on mixing, antigen is given in Chapter 15. precipitate the antigen which is then released slowly from the site of administration, thus prolonging the antigenic Superantigens exposure ‰ By activating phagocytosis: The adjuvant-antigen Superantigens are the third variety of biological class of precipitate is of larger size, thus increases the likelihood of antigens, recently described in the last decade. The unique phagocytosis. The MDP (muramyl dipeptide) component feature of superantigens is, they can activate T cells directly of tubercle bacilli can activate the macrophages directly without being processed by antigen-presenting cells ‰ By activating TH cells: Activated macrophages release (APCs). interleukin-11(IL-11) and express higher level of MHC-II; ™ The variable region of T cell receptor (v of TCR) thus promoting helper T (TH) cell activation which in turn appears to be the receptor for superantigens activates B cells to produce specific antibodies. ™ They directly bridge non-specifically between major histocompatibility complex (MHC)-II of APCs and T cells (Fig. 10.1) BIOLOGICAL CLASSES OF ANTIGENS ™ Non-specific activation of T cells leads to massive release Depending on the mechanisms of inducing antibody of cytokines known as “cytokine storm,” which include formation, antigens are classified as T cell dependent (TD) inflammatory mediators such as interferon , IL-1, IL-6, and T cell independent (TI) antigens (Table 10.1). TNF- , and TNF-  CHAPTER 10  Antigen 143 https://t.me/docinmayking Fig. 10.1: Mechanism of action of superantigens, T-dependent and T-independent antigens. Abbreviations: APCs, antigen-presenting cells; TCR, T cell receptor; MHC, major histocompatibility complex. ™ They in turn can activate B cell polyclonally, which Table 10.2: Superantigens. leads to increased secretion of non-specific antibodies Bacterial superantigen (hypergammaglobulinemia). Staphylococcal toxin: Toxic shock syndrome toxin-1 (TSST-1) Examples of Superantigens Exfoliative toxin Various products of microorganisms behave as Enterotoxins superantigens; the most important being staphylococcal Streptococcal pyrogenic exotoxin (SPE)-A and C and streptococcal toxins (Table 10.2). Mycoplasma arthritidis mitogen-I Yersinia enterocolitica Diseases Associated with Superantigens Yersinia pseudotuberculosis Viral superantigen Superantigens can cause a number of diseases. ™ Toxic shock syndrome Epstein-Barr virus associated superantigen ™ Food poisoning Cytomegalovirus associated superantigen ™ Scalded skin syndrome Rabies nucleocapsid ™ Rare conditions such as—atopic dermatitis, HIV encoded superantigen (nef- negative regulatory factor) Kawasaki syndrome, psoriasis, acute disseminated Fungal superantigen encephalomyelitis. Malassezia furfur EXPECTED QUESTIONS I. Write short notes on: c. Antigen presentation by macrophage 1. Heterophile antigens. d. Activation of complement 2. Adjuvant. 2. Which part of the bacteria is most antigenic? 3. Superantigens. a. Protein b. Carbohydrate II. Multiple Choice Questions (MCQs): c. Lipid d. Nucleic acid 1. Superantigen causes: 3. Which of the following antigen is T-dependent? a. Enhancement of phagocytosis a. Bacterial capsule b. Flagella b. Polyclonal activation of B cells c. Lipopolysaccharide d. Exotoxin Answers 1. b 2. a 3. d  CHAPTER Antibody 11 CHAPTER PREVIEW „„Structure of Antibody „„Antigenic Determinants of „„Monoclonal Antibody „„Immunoglobulin Classes Immunoglobulins „„Antibody Diversity Antibody or immunoglobulin is a specialized glycoprotein, produced from activated B cells (plasma cells) in response to an antigen, and is capable of combining with the antigen that triggered its production. https://t.me/docinmayking ™ It was found that (A. Tiselius, 1939) when the serum is subjected to electrophoresis, the serum proteins are separated into four fragments—albumin, globulin , and (Fig. 11.1). Antibodies are located in the -globulin fraction. Because they immunologically react with the antigen, they were given the name as immunoglobulin ™ Both the terms, immunoglobulin (Ig) and antibody are used interchangeably, representing the physi- ological and functional properties of the molecule respectively ™ Immunoglobulin (Ig) constitutes 20–25% of total serum proteins ™ There are five classes (or isotypes) of immunoglobulins recognized—IgG, IgA, IgM, IgD and IgE. Fig. 11.2: General structure of antibody. STRUCTURE OF ANTIBODY H and L Chain An antibody molecule is a ‘Y-shaped’ heterodimer, All four H and L chains are bound to each other by disulfide composed of four polypeptide chains (Fig. 11.2). bonds, and by noncovalent interactions, such as salt ™ Two identical light (L) chains, of molecular weight 25,000 linkages, hydrogen bonds, and hydrophobic bonds. Da each and ™ All the chains have two ends—an amino terminal end ™ Two identical heavy (H) chains each having molecular (NH3) and a carboxyl terminal end (COOH) weight 50,000 Da or more. ™ There are five classes of H chains and two classes of L chains ™ The five classes of H chains are structurally and antigenically distinct; each is designated by the Greek letters , , µ, and ™ The five classes of immunoglobulins (lgG, IgA, IgM, IgD and IgE) are classified based on the amino acid sequences of the heavy chains (Table 11.1) ™ The L chains are of two types—kappa ( ) and lambda ( ), named after Korngold and Lipari who originally described them „ In humans, 60% of the light chains are kappa and 40% Fig. 11.1: Electrophoresis of human serum proteins. are lambda type (ratio 3:2)  CHAPTER 11  Antibody 145 Table. 11.1: Type of heavy chain in each immunoglobulin class. Immunoglobulin class Heavy chain type IgG (gamma) IgA (alpha) IgM (mu) IgD (delta) IgE (epsilon) „ Both the light chains of an antibody molecule are of same type, either or , but never both. ™ L chains are composed of 214 amino acids; whereas the number of amino acids in the heavy chain varies— ranging from 446 (in chain) to 576 (in µ chain). A B Variable and Constant Regions Figs 11.3A and B: Immunoglobulin domains: A. Ig with γ, δ and α heavy chains; B. Ig with µ and ε heavy chains. Each H and L chain comprises of two regions—variable and constant region, depending upon whether the amino acid sequences of the regions show variable or uniform pattern ™ Heavy chains possess one variable domain (VH) and among different antibodies. 3 or 4 numbers of constant domain (CH): https://t.me/docinmayking „ Heavy chains , and have three constant domains- Variable Region CH1, CH2 and CH3 (Fig. 11.3A) The first 110 amino acid residues near the amino terminal „ Heavy chains µ and have four constant domains- (NH3) of both L and H chains constitute the variable CH1 to CH 4 (Fig. 11.3B). region—designated as VL and VH, respectively. It represents the antigen binding site of the antibody. Hinge Region ™ Hypervariable region: Within the variable region, there In heavy chain ( , , and ), the junction formed between CH1 are some zones (hot spots) that show relatively higher and CH2 domain constitutes the hinge region (Fig. 11.3A). variability in the amino acid sequences. Such zones are ™ This region is rich in proline and cysteine. The hinge called as hypervariable regions or complementarity region is flexible, allowing the Ig molecule to assume determining regions (CDRs). They form the antigen— different positions, thus helps the antibody in reaching binding site. There are three hot spots in the L and four towards the antigen in the H chain, respectively ™ In IgE and IgM, the and µ heavy chains do not have ™ Paratope: The site on the hypervariable regions that hinge region; instead, their constant region has an make actual contact with the epitope of an antigen is additional domain (CH 4 ) (Fig. 11.3B) called as paratope. ™ The hinge region is sensitive to various enzymatic digestions. Constant Region It constitutes the remaining part of an Ig molecule other Enzymatic Digestion than that of variable region. The length of the constant When an immunoglobulin molecule is subjected to enzy- regions is approximately 104 amino acids for light chain, matic digestion, it generates various fragments (Fig. 11.4). 330 amino acids for , and heavy chains and 440 amino ™ Papain digestion: Papain cleaves the Ig molecule at acids for µ and heavy chains. The amino acid sequence of a point above the disulfide bridge of hinge region; constant region shows uniform pattern. A single antibody resulting in three fragments each having a sedimentation molecule has two identical heavy chains and two identical coefficient of 3.5 Svedberg (S): light chains; H2L2. „ Two Fab fragments: Soluble fragments which bind to the antigen (Fab for antigen binding fragment) and Heavy and Light Chain Domains „ Fc fragment: An insoluble fraction which gets Heavy (H) and light (L) chains are further folded into crystallized in the cold (Fc for crystallizable fragment). domains, each containing about 110 amino acid residues. ™ Pepsin digestion: Pepsin cleaves the Ig molecule at a Within the domain, a loop like structure of 60 amino acids point below the disulfide bridge of hinge region; resulting is present which is formed due to an intrachain disulfide in formation of: bond. The number of domains in each chain varies: „ One F (ab’) 2 fragment: A fragment having a ™ Light chain contains one variable domain (VL) and one sedimentation coefficient of 5S; composed of two Fab constant domain (CL) subunits bound together 146 SECTION 2  Immunology IMMUNOGLOBULIN CLASSES Based on five types of heavy chains, there are five classes of immunoglobulins (lgG, IgA, IgM, IgD and IgE). Each class can also exist as two types due to presence of different light chain type—kappa or lambda. IgG and IgA are further divided into subclasses (four for IgG and two for IgA) due to minor differences in amino acid sequences in constant region of heavy chains. Important properties of different Ig classes are summarized in Table 11.2. Immunoglobulin G (IgG) It constitutes about 70–80% of total Ig in the body. ™ Among all Ig, IgG has maximum daily production, longest half-life of 23 days and highest serum concentration ™ IgG has four subclasses: IgG1, IgG2, IgG3 and IgG4; all differ from each other in the amino acid sequences of the constant region of their -heavy chain ™ The subclasses vary in their biological functions, length of hinge region and number of disulfide bridges. IgG3 https://t.me/docinmayking has longest hinge region with 11 interchain disulfide Fig. 11.4: Enzymatic digestion of immunoglobulin. bonds. „ Many smaller fragments: Due to digestion of Fc portion by pepsin into smaller fragments. Functions of IgG ™ Mercaptoethanol reduction of Ig molecule—generates ™ IgG can cross placenta; hence provide immunity to the four fragments (two H and two L chains) as it cleaves only fetus and newborn. Among subclasses, IgG2 has the disulfide bonds sparing the peptide bonds. poorest ability to cross placenta ™ Complement fixing: Fc region of IgG can bind to FUNCTIONS OF IMMUNOGLOBULINS complement factors; thus activates the classical pathway of complement system. The complement-fixing ability Antigen Binding (by Fab Region) of subclasses varies—IgG3> IgG1> IgG2. IgG4 does not Binding to the antigen is the primary function of an fix complements antibody which can result in protection of the host. ™ Phagocytosis: IgG1 and IgG3 bind to Fc receptors present ™ The Fab fragment bears the variable region and is on phagocytes (macrophages, neutrophils) with high involved in interaction with the antigen affinity and enhance the phagocytosis (opsonization) ™ The valency of an antibody refers to the number of Fab of antigen bound to them. IgG2 has an extremely low regions it possesses. Thus, a simple monomeric antibody affinity for Fc receptors of phagocytes molecule has a valency of two. ™ It mediates precipitation and neutralization reactions ™ IgG plays a major role in neutralization of toxins as it can Effector Functions (by Fc Region) easily diffuse into extravascular space Most of the times, the binding of an antibody to its antigen ™ IgG is raised after a long time following infection and does not result in any direct biological effect. Rather, variety represents chronic or past infection (recovery) of secondary “effector functions” are produced; mediated by ™ Coagglutination: IgG subclasses (except IgG3) mediate Fc region of the antibody. These effector functions include: coagglutination reaction by binding to protein-A of S. ™ Fixation of complement: Antibody coating the target aureus (refer Chapter 12). cell binds to complement through its Fc receptor which leads to complement mediated lysis of the target cell Immunoglobulin M (IgM) ™ Binding to various cell types: Phagocytes, lymphocytes, Among all immunoglobulins, IgM has highest molecular platelets, mast cells, NK cell, eosinophils and basophils weight, and maximum sedimentation coefficient (19S). It bear Fc receptors (FcR) that bind to Fc region of is present only in intravascular compartment, not in body immunoglobulins. This binding can activate the cells fluids or secretions. to perform some biological functions (described with IgM exists in both monomeric and pentameric forms: individual immunoglobulins and also in Chapter 15). ™ When present as membrane-bound antibody on B cells, Some immunoglobulins (e.g. IgG) also bind to receptors it exists in monomeric form on placental trophoblasts, which results in transfer of IgG ™ When present in secreted form, it is pentameric in nature; across the placenta. i.e. five IgM monomeric units are joined with each other  CHAPTER 11  Antibody 147 Table 11.2: Properties of various immunoglobulins. Property IgG IgA IgM IgD IgE Usual form Monomer Monomer, dimer Monomer, pentamer Monomer Monomer Valency 2 2 or 4 2 or 10 2 2 Other chains None J chain, secretory J chain None None component Subclasses G1, G2, G3, G4 A1, A2 None None None Molecular weight (kDa) 150 150–600 900 150 190 Serum level (mg/mL) 9.5–12.5 IgA 1–3.0 1.5 0.03 0.0003 IgA 2–0.5 % of total serum Ig 75–85% 10–15% 5–10% 0.3% 0.019% Half-life, days 23* 6 5 3 2.5 Daily production (mg/kg) 34 24 3.3 0.4 0.0023 Intravascular distribution (%) 45 42 80 75 50 Sedimentation coefficient (S) 7 7 19 7 8 Complement activation Classical ++ (IgG3>1>2) – +++ – – Alternative – + – – – https://t.me/docinmayking Binds to Fc receptors of phagocytes ++ – ? ** – – Placental transfer Yes (except IgG2) – – – – Mediates coagglutination Yes (except IgG3) – – – – Mucosal transport – Yes – – – Mast cell degranulation – – – – yes Marker for B cells – – + + – Heat stability + + + + – *Half-life of IgG3 is 8 days; **?- Questionable ™ It acts as an opsonin; binds to antigen which is then easily recognized and removed. IgM is 500–1000 times more potent in opsonization than IgG ™ Fetal immunity: It is the first antibody to be synthesized in fetal life (20 weeks); thus provides immunity to the fetus. Presence of IgM in fetus or newborn indicates intrauterine infection (as it cannot cross placenta), and its detection is useful in the diagnosis of congenital infections ™ Protection against intravascular organisms: IgM being A B intravascular, is responsible for protection against blood invasion by microorganisms. IgM deficiency is often Figs 11.5A and B: A. Pentameric IgM; B. Dimeric IgA. associated with septicemia (by J chain) to form a complete IgM pentamer having 10 ™ Mediates agglutination: IgM is about 20 times more Fab regions and 10 valencies (Fig. 11.5A). effective in bacterial agglutination than IgG. Functions of IgM Immunoglobulin A (IgA) ™ Acute infection: IgM is the first antibody to be produced IgA is the second most abundant class of Ig next to IgG, following an infection; represents acute or recent constituting about 10–15% of total serum Ig. It exists in both infection. It is also called as primary immune response monomeric and dimeric forms (Fig. 11.5B). antibody ™ Complement fixing: It is the most potent activator Serum IgA of classical complement pathway due to multiple ™ IgA in serum is predominantly in monomeric form complement binding sites (5 Fc regions) present in IgM ™ Functions: Serum IgA interacts with the Fc receptors pentamer expressed on immune effector cells, to initiate various ™ It is also present on B cell surface in monomeric form and functions such as antibody-dependent cell-mediated serves as B cell receptor for antigen binding cytotoxicity (ADCC), degranulation of immune cells, etc. 148 SECTION 2  Immunology Secretory IgA Functions of IgE Secretory IgA is dimeric in nature; the two IgA monomeric ™ IgE is highly potent and mediate type I hypersensitivity units are joined by J chain. In addition, there is another reactions by binding to the mast cells causing degranula- joining segment present between two IgA molecules called tion. IgE response is seen in various allergic conditions, secretory component. such as asthma, anaphylaxis, hay fever, etc. (Described ™ Location: Secretory IgA is the predominant antibody in detail in Chapter 16) found in body secretions like milk, saliva, tears, intestinal ™ IgE is elevated in helminthic infections. By coating on and respiratory tract mucosal secretions the surface of eosinophils, IgE stimulates the release of ™ The secretory component is derived from poly-Ig the mediators on to the surface of helminths by a process receptor present on the serosal surfaces of the epithelial known as antibody mediated cellular cytotoxicity or cells ADCC (Chapter 15). ™ Function: The secretory IgA mediates local or muco- sal immunity; provides protection against pathogens Immunoglobulin D (IgD) by cross-linking bigger antigens with multiple epit- IgD is found as membrane Ig on the surface of B cells and opes and preventing their entry through the mucosal acts as a B cell receptor along with IgM. It has the highest surface carbohydrate content among all the immunoglobulin. No „ It is effective against bacteria like Salmonella, Vibrio, other function of IgD is known so far. Neisseria, and viruses like polio and influenza „ Breast milk is rich in secretory IgA and provides good ANTIGENIC DETERMINANTS OF protection to the immunologically immature infant’s IMMUNOGLOBULINS https://t.me/docinmayking gut. Since antibodies are glycoproteins, they can themselves Secretory IgA function as potent immunogens, having a number of The dimeric secretory IgA is synthesized by plasma cells antigenic determinants which can induce antibody situated near mucosal epithelium. The J chain is also produced responses in hosts other than the parent host. It is observed in the same cell. that the entire Ig molecule is not immunogenic, but it Whereas, the secretory component is synthesized by the contains antigenic determinants at specific sites. Based on mucosal epithelial cells. the location of antigenic determinants, the Ig molecules ‰ It is derived from the poly-Ig receptor present on the are divided into isotypes, idiotypes and allotypes (Fig. basolateral surfaces of the epithelial cells 11.6). ‰ It helps the dimeric IgA to cross the epithelial surface to reach the lumen Isotypes ‰ It also protects IgA from denaturation by bacterial proteases produced by intestinal flora. The five classes of Ig (lgG, IgA, IgM, IgD and IgE) and their subclasses are called as isotypes; they vary from each other in the amino acid sequences of the constant region Subclasses of IgA of their heavy chains. Such variation is called as isotypic Depending upon the amino acid sequences in the con- variation. stant region of heavy chain, IgA exists as two isotypes: ™ Isotypes that are present in all members of a given species 1. IgA1 is the dominant subclass in serum. Serum IgA are similar in nature comprises of ~90% IgA1 and 10% IgA2 ™ Hence, antibody against isotypes can be produced by 2. IgA2 is present in higher concentration in secretions than injecting the Ig from one species into another. in serum (ranging from 10% to 20% in nasal and male genital secretions, 40% in saliva, to 60% in colonic and female genital secretions) „ IgA2 lacks the disulfide bonds between the heavy and light chains „ Polysaccharide antigens tend to induce more IgA2 synthesis than protein antigens. Immunoglobulin E (IgE) Among all Ig, IgE is having the lowest serum concentration, shortest half-life and minimum daily production. It is also the only heat labile antibody (inactivated at 56°C in one hour). It has affinity for the surface of tissue cells (mainly mast cells) of the same species (homocytotropism). It is mainly extravascular in distribution. Fig. 11.6: Antigenic determinants of immunoglobulins.  CHAPTER 11  Antibody 149 Idiotypes chains (Bence Jones proteins) which are accumulated The unique amino acid sequence present in paratope in patient’s serum and excreted in urine ™ Such proteins have a unique property of getting region (in VH and VL regions) of one member of a species acts as antigenic determinant to other members of the coagulated at 50°C and redissolving again at 70°C. same species. Other abnormal immunoglobulins include: ™ Waldenstrom’s Macroglobulinemia: It is a B cell ™ Such antigenic determinants are called as idiotopes and the sum total of idiotopes on an Ig molecule constitutes lymphoma, producing excess IgM ™ Heavy chain disease: It is characterized by an excessive its idiotypes (Fig. 11.6) ™ Such variation between immunoglobulins due to production of heavy chains that are short and truncated ™ Cryoglobulinemia: Seen in multiple myeloma and differences in the amino acid sequences of the variable hepatitis C infection. region is called as idiotypic variation ™ Idiotypes in an individual arise continuously from mutations (somatic hypermutations) in the genes of MONOCLONAL ANTIBODY variable region. Hence, idiotypes may act as foreign to the Monoclonal antibodies (mAb) are defined as the antibodies host itself; however, do not evoke autoimmune response derived from a single clone of plasma cell; all having the because they are present in small numbers. same antigen specificity, i.e. produced against a single epitope of an antigen. Allotypes The antigenic determinants present in the isotype genes in Polyclonal vs Monoclonal Nature of Antibody https://t.me/docinmayking the constant region of H and L chains, encoded by multiple When an antigen having multiple epitopes enters the body, alleles are called as allotypes. each epitope may stimulate one clone of B cells producing ™ Although all members of a species inherit the same set one type of antibody. Hence the resultant antibody mixture of isotype genes, multiple alleles exist for some of the present in serum is said to be polyclonal, i.e. contains allele genes mixture of antibodies derived from different clones of ™ Hence, allotypes are present in the constant region of B cells. Ig molecules of the same class, in some, but not all, However, when only one clone of B cell is stimulated members of a species by a single epitope of an antigen and then is allowed to ™ The sum of the individual allotypic determinants proliferate and produce antibodies; such antibodies are displayed by an antibody determines its allotype referred to as monoclonal antibodies (mAb). ™ Allotypes differ in sequence of 1–4 amino acid from one another Production of mAb (Hybridoma Technique) ™ Allotype systems: To date, three systems of allotypic Monoclonal antibodies are produced by Hybridoma markers have been characterized for humans: technique, developed by G Kohler and C Milstein (1975), „ For kappa light chain (Km system)—has three Km for which they were awarded Nobel Prize in 1984. allotypes Principle „ For heavy chain (Gm system)—has 25 Gm types „ For heavy chain (Am system). A clone of B cell stimulated against a single epitope of anti- Antibody to allotype determinants can be produced gen is fused with an immortal cell, e.g. myeloma cell (capa- by injecting antibodies containing these determinants ble of multiplying indefinitely) to produce a hybridoma cell. from one member to another within a given species. This hybridoma cell has two unique properties: Anti allotype specific antibodies may also be developed 1. Produces monoclonal antibody of same antigen following blood transfusion or by maternal passage of IgG specificity (due to B cell component) into the fetus. 2. Multiplies indefinitely producing clone of identical cells (due to immortal myeloma cell component). ABNORMAL IMMUNOGLOBULINS Procedure In addition to the five classes of normal antibodies, other The steps of hybridoma technique are as follows (Fig. 11.7): structurally similar proteins are seen in sera of patients and ™ Mouse splenic B cells: The mouse is injected with sometimes even in healthy individuals. an antigen containing the desired epitope. After an interval, the mouse splenic B cells are obtained which Bence Jones Proteins are activated against the epitope of the antigen injected They are produced in a neoplastic condition of plasma cells ™ Myeloma cells are used as a source of immortal cells. called multiple myeloma. They are cancerous plasma cells. They closely resemble ™ This condition is also called as light chain disease as mouse B cells; hence are compatible for fusion. However, the cancerous plasma cells produce excess of light myeloma cells also have the capacity to produce their 150 SECTION 2  Immunology „ Aminopterin blocks the de novo pathway so that the cell has to perform the salvage pathway to synthesize purines for its survival „ Salvage pathway requires two important enzymes- HGPRT (hypoxanthine guanine phosphoribosyl- transferase) and thymidine kinase „ So any cell (e.g. myeloma cell) that lacks HGPRT cannot grow on HAT medium. ™ Fate of three types of cells on HAT media: 1. Unfused splenic B cells: They can grow, but do not survive long as they are not immortal 2. Unfused myeloma cells: They cannot grow as they lack HGPRT enzyme to perform the salvage pathway of purine synthesis 3. Hybridoma cells: They can grow and survive long. ™ Selection of individual hybridoma cells: If the original antigen used has multiple epitopes, many B cells would fuse with myeloma cells to produce a mixture of hybridoma cells each having specificity for one epitope https://t.me/docinmayking „ The medium containing hybridoma cells is then diluted into multi-well plates to such an extent that each well contains only one cell „ The hybridoma cells producing the desired mAb are selected by radioimmunoassay or ELISA techniques using the specific antigen fragments, and are selectively proliferated. ™ Maintenance of mAb: The selected hybridoma cells can be maintained in two ways: 1. Hybridoma cell is cultured to generate a clone of identical cells; producing pure form of monoclonal antibodies at a concentration of 10–60 µg/mL 2. Alternatively the desired hybridoma cell is injected into the peritoneal cavity of mouse where it can Fig. 11.7: Hybridoma technology. multiply and produce mAb in ascitic fluid at a Abbreviation: HGPRT, hypoxanthine guanine phosphoribosyl transferase. concentration of 1–10 mg/mL. Such mAb obtained from mouse ascitic fluid and serum may not be in own antibodies. Hence myeloma cells are genetically pure form, is often mixed with other antibodies; modified with two mutations (double mutated myeloma hence, it is purified by chromatography or by cells), so that they lose the ability to produce their own immunoprecipitation test. antibody but retain immortal property ™ Fusion: The mouse splenic B cells and mutated myelo- Types of Monoclonal Antibodies ma cells are fused in polyethylene glycol broth. In the The above mentioned procedure would yield mAb whose reaction chamber, three types of cells will be generated: 100% amino acids are mouse derived. The problem of 1. Unfused myeloma cells mouse mAb is that, the mouse proteins being foreign; can 2. Unfused mouse splenic B cells induce immune response in humans producing human 3. Fused hybridoma cells. anti-mouse antibodies (HAMA); that in turn eliminate ™ Purification (by subculturing on HAT media): The next the mAb faster from the body. Hence mouse derived step is to remove the unwanted unfused cells and to monoclonal antibodies are not the best for human use. propagate the clone of hybridoma cells. This is carried Since the discovery of hybridoma technique, various out by subculturing the cells in reaction chamber onto modifications have been attempted to produce mAb by a special medium called HAT medium recombining human and mouse proteins (Fig. 11.8). ™ HAT medium: It contains hypoxanthine, aminopterin ™ Mouse mAb: It contains 100% mouse derived proteins and thymidine ™ Chimeric mAb: It is prepared by recombination of „ Purine synthesis in mammalian cell (e.g. splenic B 34% mouse proteins (variable region) and 66% human cell) occurs by either de novo or salvage pathways proteins (constant region)  CHAPTER 11  Antibody 151 ™ Therapeutic use: Monoclonal antibodies are used in the treatment of various inflammatory and allergic diseases and cancers (Table 11.3) ™ Used as immunotoxin: mAb conjugated with bacterial/chemical toxins (e.g. diphtheria toxin) can be used to kill the target cells such as cancer cells. Here, mAb against surface receptors helps in binding to the target cells and the toxin helps in target cell killing ™ Used as enzymes: Abzyme is a monoclonal antibody with catalytic activity. ANTIBODY DIVERSITY Antibody diversity is a mechanism by virtue of which human immune system is capable of producing vast number of antibodies (108 or even more) corresponding to various epitopes of different antigens. There are several postulates which explain the mechanism of antibody diversity. ™ Multiple chromosomes: Ig chains are coded on different chromosomes; H chain on chromosome 14, whereas light chain kappa and lambda on chromosome 2 and 22 respectively Fig. 11.8: Types of monoclonal antibodies. ™ Multiple genes exist for each segment (VH, CH, VL and CL) of Ig chain. For example, there are 51 VH genes known to exist in https://t.me/docinmayking ™ Humanized mAb: Here, only the antigen binding site nature and out of which any one gene would code for VH chain (i.e. CDR—complementarity determining region) is of an Ig. In this way, there are many possible combinations of joining of the Ig gene segments. mouse derived (10%) and the remaining part of mAb is human derived Table 11.3: Therapeutic uses of monoclonal antibodies. ™ Human mAb: It contains 100% human derived amino Monoclonal Targeted against Used in treatment acids. It is the best accepted mAb in humans. antibody of Suppress immune system Applications of Monoclonal Antibodies Adalimumab TNF-α Rheumatoid arthritis ™ Isolation and purification: Monoclonal antibodies can be used and infliximab Crohn’s disease to purify individual molecule from a mixture when they are Omalizumab IgE Asthma present in low concentration, e.g. interferon and coagulation Daclizumab IL-2 receptor Rejection of kidney factor VIII transplants ™ Muromonab CD3 Identification of cells and clones: For example TH and TC cells are identified by using anti-CD4 and anti-CD8 mAb Anticancer ™ Diagnostic reagents: The widest application of mAb is Trastuzumab HER-2 Breast cancer detection of antigen. The antigen detection kits employ various Rituximab CD20 Lymphoma mAb tagged with detection molecules, such as fluorescent Inhibit angiogenesis dye or enzyme to detect the specific antigens in the clinical Bevacizumab VEGF (vascular endothelial Colorectal cancers specimen by using various formats like ELISA, rapid tests, etc. growth factor) Examples include detection of hepatitis B surface antigen, Abciximab Platelet receptor GpIIb/IIIa Coronary artery serogrouping of streptococci, etc. disease ™ Monitoring of proteins and drug levels in serum ™ Passive immunity: For post-exposure prophylaxis against Note: ‰ Mouse mAb ends with suffix ‘umab or onab’ various infections, mAb targeting specific antigens of the ‰ Chimeric mAb ends with suffix ‘ximab’ infecting organism can be administered. Examples include— ‰ Humanized mAb ends with suffix ‘zumab’ immunoglobulins against hepatitis B, rabies, and tetanus ‰ Human mAb ends with suffix ‘mumab’ EXPECTED QUESTIONS I. Write essay on: 2. What is the total valancies of IgM? 1. Define antibody. Describe in detail about the structure a. 10 b. 5 c. 2 d. 1 and functions of various types of antibodies. 3. Which antibody is elevated in acute infection? 2. Monoclonal antibodies and their applications. a. IgA b. IgG c. IgE d. IgM II. Multiple Choice Questions (MCQs): 4. Which antibody mediates mucosal immunity? 1. Which antibody crosses placenta? a. IgA b. IgG c. IgE d. IgM a. IgA b. IgG c. IgE d. IgM Answers 1. b 2. a 3. d 4. a  CHAPTER Antigen–Antibody Reaction 12 CHAPTER PREVIEW „„General Properties „„Newer Techniques „Chemiluminescence „„Conventional Immunoassays „ELISA „Western Blot „Precipitation Reaction „Enzyme-linked Fluorescent Assay „Rapid Tests „Agglutination Reaction „Immunofluorescence Assay The antigen–antibody reaction is a bimolecular association Avidity where the antigen and antibody combine with each other It is a term used to describe the affinities of all the binding https://t.me/docinmayking specifically and in an observable manner similar to an sites when multivalent antibody reacts with a complex enzyme-substrate interaction, the only difference is, it does antigen carrying multiple epitopes. not lead to an irreversible alteration in either antibody or ™ The total strength (i.e. avidity) would be much higher in antigen. than the individual affinity at each binding site, but lower than the sum of all affinities. This difference is primarily GENERAL PROPERTIES OF ANTIGEN– due to geometry of Ag-Ab binding ANTIBODY REACTIONS ™ The geometry of the multivalent antibody gets stretched when it reacts with a complex antigen, as it has to reach Antigen (Ag)–antibody (Ab) reactions are characterized by and accommodate all the epitopes, thus resulting in less the following general properties: optimal binding interactions Specific ™ Avidity is a better indicator of strength of an antigen- antibody reaction. Avidity of an antibody can compensate Ag-Ab reaction involves specific interaction between for its low affinity. For example, IgM has a low affinity epitope of an antigen with the corresponding paratope than IgG, but it is multivalent (10 valencies), therefore has of its homologous antibody. Exception is the cross reac- a much higher avidity. Hence, it can bind to an antigen tions which may occur due to sharing of epitopes among more effectively than IgG different antigens. In such cases, antibody against one an- ™ Avidity increases with time: Though IgG has a low avidity tigen can cross react with a similar epitope of a different initially, IgG produced in later part of infection will have antigen. stronger avidity. This property is used in IgG avidity test; Noncovalent Interactions described later in this chapter. The union of antigen and antibody requires formation of Diagnostic Use large number of non-covalent interactions between them Because Ag-Ab reactions are specific and observable, they such as: are extensively used in the laboratories for the diagnosis of ™ Hydrogen bonds infectious diseases. The diagnostic tests based on Ag-Ab ™ Electrostatic interactions reactions are called as immunoassays. Most immunoassays ™ Hydrophobic interactions are also called serological tests as they are performed using ™ Van der Waals forces. serum samples. However, other samples can also be used Strength such as urine, CSF, etc. Immunoassays can be broadly categorized into two types: The strength or the firmness of the association is influenced 1. Antigen detection assays: Detect antigens in patient’s by the affinity and avidity of the antigen–antibody interac- sample by employing specific antibody tion. 2. Antibody detection assays: Detect antibodies in patient’s sample by employing specific antigen. Affinity It refers to the sum total of noncovalent interactions between Qualitative vs Quantitative Immunoassays a single epitope of an antigen with its corresponding Immunoassays can be performed by both qualitative and paratope present on antibody. quantitative methods.  CHAPTER 12  Antigen–Antibody Reaction 153 Qualitative Assays Here, the undiluted specimen containing the antibody is directly mixed with the suspension of antigen or vice versa. The result is read as ‘positive’ or ‘negative’ based on presence or absence of antigen or antibody in the clinical specimen. The exact amount of antigen or antibody present in the specimen cannot be estimated. Quantitative Assays A B C When the qualitative test turns positive, the exact amount of antibody in serum can be estimated by serial dilution of the patient’s serum and mixing each dilution of the serum with a known quantity of antigen. The measurement of antibody is expressed in terms of titer. Figs 12.1A to C: A. Prozone; B. Zone of equivalence; C. Postzone. ™ The antibody titer of a serum is the highest dilution that Contd... shows an observable reaction with the antigen ™ Antigen titer can also be measured in the sera in similar Lattice hypothesis was described first for precipitation reaction, fashion by testing the series of diluted sera against known but it also holds true for agglutination and other techniques of quantity of antibody. Ag-Ab reactions. The problem with qualitative test is that if the number The prozone phenomenon is of great importance in clinical https://t.me/docinmayking of antigen or antibody molecules in the reaction are serology, as sera rich in high titer of antibody may sometimes give a false-negative result, unless serial dilutions of sera are disproportionate to each other and if either antigen or tested. antibody are present in higher quantity, then the antigen antibody reaction does not take place optimally and often the result turns negative (false-negative). To rule out a Evaluation of Immunoassays false negative result, it is ideal to test a series of diluted Evaluation of the performance of any diagnostic test in- sera (quantitative test), instead of just testing once on an cluding immunoassays can be done by calculating various undiluted serum. Quantitative tests are more reliable as they statistical measures. Among all, sensitivity and specificity can differentiate between true negative and false-negative are the two most important statistical parameters. results. This can be explained by Marrack’s Lattice hypothesis. Sensitivity is defined as ability of a test to identify correctly Marrack’s Lattice Hypothesis all those who have the disease, i.e. true-positives. When the sera containing antibody is serially diluted (in True positives normal saline), gradually the antibody level decreases. When Sensitivity is calculated as = True positives + False negatives a fixed quantity of antigen is added to such a set of test tubes Specificity is defined as ability of a test to identify correctly containing serially diluted sera, then it is observed that the all those who do not have disease, i.e. true negatives. Ag-Ab reaction occurs at its best only in the middle test tubes where the amount of antigen and antibody are equivalent to True negatives Specificity is calculated as = each other (zone of equivalence). The Ag-Ab reaction is weak or True negatives + False positives fails to occur when the number of antigen and antibodies are not proportionate to each other (Figs 12.1A to C). TYPES OF ANTIGEN–ANTIBODY REACTIONS ‰ In the earlier test tubes, antibodies are excess, hence the Ag-Ab reaction does not occur: This is called as prozone The antigen–antibody reactions used in diagnostic phenomenon laboratories are based on various techniques which are ‰ In the later test tubes, antigen is excess, hence the Ag- broadly classified as conventional techniques and newer Ab reaction fails to occur: This is called as postzone techniques (Table 12.1). phenomenon. Marrack (1934) proposed the lattice hypothesis to explain this mechanism. According to this concept the multivalent CONVENTIONAL IMMUNOASSAYS antigens combine with bivalent antibodies in varying proportions, depending on the antigen antibody ratio in the PRECIPITATION REACTION reacting mixture (Figs 12.1A to C). ‰ Ag-Ab reaction optimally occurs when a large lattice is Definition formed consisting of alternating antigen and antibody When a soluble antigen reacts with its antibody in the molecules. This is possible only in the