Antigen and Antibody: Structure and Function PDF

ROSS UNIVERSITY SCHOOL OF MEDICINE STUDENT OUTCOMES OVERVIEW ANTIGEN AND ANTIBODY: STRUCTURE AND FUNCTION Raymond F Adebiyi Professor Immunology and Medical Microbiology Learning Objectives 1. 2. 3. 4. 5. 6. 7. 8. Define and differentiate between antigen and immunogen. Name the necessary char...

ROSS UNIVERSITY SCHOOL OF MEDICINE STUDENT OUTCOMES OVERVIEW ANTIGEN AND ANTIBODY: STRUCTURE AND FUNCTION Raymond F Adebiyi Professor Immunology and Medical Microbiology Learning Objectives 1. 2. 3. 4. 5. 6. 7. 8. Define and differentiate between antigen and immunogen. Name the necessary characteristics that enable an antigen to drive an immune response. Define adjuvant. Define hapten. Describe what happens between the time an antigen enters the body and the appearance of free antibody What is an epitope? Describe the basic structure of an antibody molecule and explain the functions of its component parts. List the products of papain and pepsin digestion of IgG and discuss the functions and significance of the products. Name the five isotpyes (classes of antibodies). What is/are the structure(s) of the secreted forms of each of the isotypes? Describe the unique attributes of each class. List the tissue distribution and the biological functions (effector functions) of each class. Any Questions? [email protected] Office Hours https://atge.webex.com/meet/radebiyi The Immune System • Function – Detect potentially hazardous materials then – Eliminate or inactivate them • To perform that function, it must – Be able to recognize these materials, and – Distinguish them from “self” ANTIGEN Antigen • Modern definition: – A molecule that is recognized by the immune system – A substance that can induce an immune response, usually foreign - note that the immune response is not limited to antibody production; may be humoral or cellular, innate or adaptive Properties of Antigens • Size – at least 6,000-10,000 daltons (6-10 kd) • Complex – Proteins are the best antigens because of their complex nature – Polysaccharides also make good antigens – Nucleic acids and phospholipids are weak antigens – Small molecules (hormones, lipids, simple sugars) usually must be conjugated to a larger macromolecule to be antigenic Properties of Antigens • Foreignness – if it does not look foreign, it is not a good antigen • Solubility – molecules that are easily solubilized are better antigens – Insoluble molecules tend to pass through body unrecognized Antigens vs. Immunogens • All immunogens immunogens are antigens antigens antigens • BUT, not all antigens • Antigens immunogens are immunogens that provoke an immune response are termed immunogens Factors influencing immunogenicity • Dose – Very small quantities may get rapidly cleared – Very large quantities may inhibit response • Route – Entry of host for most pathogens by crossing mucosal surface  GI: most are rapidly degraded and do not get much chance to interact with immune cells – Those that enter bloodstream can remain intact and have more potential to drive immune response Adjuvants can impact immunogenicity • Adjuvants: chemical substances that enhance the immune response to an antigen • Thought to: – Prolong the persistence of the antigen – Stimulate or modulate immune cells – Enhancing macrophage function Haptens • Haptens – small molecules that, on their own, are not immunogenic – can bind antibody – generally only have one epitope • To make a hapten immunogenic, it must be bound to a protein carrier molecule to form a conjugate Epitopes • Epitope (antigenic determinants): portion of antigen that binds the antigen recognition molecule • Number of epitopes = valence of the antigen – Part of the antigen recognized by antibody – Peptide recognized by the T cell receptor PHASES OF ANTIGEN ELIMINATION A B E C D PHASES OF ANTIGEN ELIMINATION 1. Equilibration Phase – distribution of antigen between vascular and the extravascular compartments 2. Catabolic Phase - Elimination of antigen by innate cells; processing and presentation of antigen 3. Immune Elimination Phase – The formation of antigenantibody immune complex and removal of complex 4. The appearance of free antibody in serum Note that antibody production starts at the end of Phase 2 (C) but there is no free antibody during the formation of immune complex in Phase 3 (C-D) ANTIBODY A B cells’ job is to make antibodies Antibody = Immunoglobulin = B cell receptor (when surface-bound) Antibody: Basic Structure ANTIBODY STRUCTURE Antibodies trigger phagocytosis Fc Receptors allow recognition of the Fc portion of antibodies How the Immune System Works 4th ed. Wiley Blackwell 2012 THE GENERATION OF ANTIBODY DIVERSITY LEVEL 1: -Combinatorial Diversity- Antigen recognition site is created through gene segment rearrangements/recombinations mediated by RAG-1 and RAG-2 From “How the Immune System Works” by L. Sompayrac , 2012 p.5 GENERATION OF COMBINATORIAL DIVERSITY LEVEL 2: -Junctional Diversity- The formation of the junction between gene segments involves DNA cleavage and the addition or deletion of nucleotides to create a joint. This random addition by the enzyme terminal deoxynuceotidyl transferase TdT, or random deletion by exonuclease, results in a further level of variability or junctional diversity. GENERATION OF JUNCTIONAL DIVERSITY MEDIATED BY TERMINAL DEOXYNUCLEOTIDYL TRANSFRERASE (TdT) Diverse specificity is reflected in the degree of amino acid variability in the hypervariable, HV regions, also called the complementarity determinant region (CDR) and framework regions (FR) within the variable-region Proteolytic enzyme products tell us about the structure of the antibody molecule Papain: IgG  2Fab + Fc Pepsin: IgG  (Fab’)2 + Fc’ SIGNIFICANCE OF ENZYMATIC CLEAVAGE OF IgG Ability to cross the placenta -Role of FcR Fab cannot cross  Fc can cross  Fab cannot cross  Fc cannot cross CHARACTERISTICS OF THE CONSTANT REGION OF IMMUNOGLOBULIN  constant sequence  crystalizable  carboxyl terminal  cell binding  complement binding  complement activating Idiotypes, Allotypes & Isotypes Isotypic difference Immunobiology ( Garland Science 2005) Allotypic difference Idiotype difference Idiotypes, Allotypes & Isotypes Isotypic difference Allotypic difference Idiotype difference Immunobiology ( Garland Science 2005) Idiotypic variation – variations in variable region, particularly hypervariableregion Antibodies - Structure IDIOTYPE: Defines the specificity Which antigen will it bind? ISOTYPE: Defines the function How does the antibody contribute to the immune response? Idiotypes, Allotypes & Isotypes Isotypic difference Allotypic difference Immunobiology ( Garland Science 2005) Allotypic variation – genetic differences involving different alleles Idiotype difference Idiotypes, Allotypes & Isotypes Isotypic difference Immunobiology ( Garland Science 2005) Allotypic difference Idiotype difference Isotypes IgM • First antibody secreted in immune response • Found on surface of naïve B cells • Secreted as a pentamer – 5 identical units – Bound by joining “J” chain – 10 antigen binding sites • Readily activates classical complement IgM is strong activator of classical complement pathway IgG • Second antibody to be secreted • Secreted in large amounts • Most abundant isotype in blood and lymph • Secreted as a monomer • Functions – Activate classical complement – Opsonization of pathogens • Triggering phagocytosis • Triggering Antibody-Dependent Cell Cytotoxicity (ADCC) – Neutralization IgA • Secreted as a monomer and as a dimer • Dimeric form contains J chain and secretory component – Secretory component facilitates transport across epithelium into secretions of the mucosal system (process called trancytosis) • Most predominant isotype produced in gut • Main function: neutralization IgE • Secreted as monomer • Binds to high affinity Fc receptors on mast cells, eosinophils and basophils • Efficiently triggers degranulation • Important in allergy and response to helminthes IgD • Expressed on the surface of naïve B cells • Secreted form has no known function Antibody isotype tissue distribution • IgM found only in blood • IgG found in blood, tissues and can cross placenta • IgA found in • Lumen of gut • In secretions (saliva, tears, milk) • On mucous membranes (respiratory and GI/GU) • IgE found bound in Fc receptors on mast cells in tissues Microbiology & Immunology Antigen & Antibody Dr. Raymond F Adebiyi Antigen and Antibody: Structure and Function LearningObjectives: 1. Defineanddifferentiatebetweenantigen,immunogen,andhapten. 2. Name somecharacteristics thatenablean antigentodriveandimmuneresponse.Namesome properties that impact the immunogenicity of an antigen. Define “adjuvant” and describe how adjuvants influence immunogenicity. 3. Whatisanepitope?Contrastlinearandconformationalepitopes. 4. Describetheantigeneliminationcurve.Explaintheprocessesthatprecedetheappearanceof free antibody. 5. Describethebasicstructureofanantibodymoleculeandexplainthefunctionsofitscomponent parts. 6. Explainhowthegenerationofantibodydiversityis achieved. 7. ListtheproductsofpapainandpepsindigestionofIgGanddiscussthefunctionsand significance of the products. 8. Namethefiveisotypes(classesofantibodies).Whatis/arethestructure(s)ofthesecreted formsofeach ofthe isotypes?Describe the unique attributesof each class. List thetissue distribution and the biological functions (effector functions) of each class. Recommended Reading: Helbert, M., 2017, Immunology for Medical Students, 3rd edition. Mosby International, Chapter 4, pp. 18 – 24 1 Microbiology & Immunology Antigen & Antibody Antigens & Immunogens– General Properties Dr. Raymond F Adebiyi The function of the immune system is to detect potentially dangerous materials in the body, and to drive the production and activation of cells or cell products that destroy or inactivate such materials. The efficacy of this function depends on two fundamental principles (1) (2) recognition and Self-discrimination. In order to perform its protective function, the immune system must be capable of recognizing hazardous materials and must differentiate these from “self” materials. All materials, self or non-self, are composed of chemical substances linked in various configurations. The molecules recognized by the immune system are called antigens and they must possess the following characteristics to provoke an immune response: Size: The typical antigen is a molecule with a minimum molecular mass of about 10,000 Daltons. Smaller molecules tend to be weakly antigenic or haptenic. Complexity: Protein molecules are the most antigenic on the basis of their complex nature. Carbohydrates, lipids and nucleic acids, which tend to contain repeating subunits, are poorly antigenic. Additionally, nucleic acids and phospholipids are highly conserved across biologic systems and tend to need special conditions to stimulate an immune response. Hormones, metabolic by-products, lipids, simple sugars, and other small molecules are antigenic, but typically need to be conjugated to a larger macromolecule to stimulate an immune response. Foreignness: Fundamentally, the immune system is programmed to react against “nonself”. Therefore, molecules that are not recognized as foreign are not antigenic. Solubility: It is also recognized that molecules that are soluble or easily solubilized are better antigens than those that are insoluble. Insoluble or nondegradable materials tend to pass through the body virtually unchanged. Not all antigens recognized by immune cells induce an immune response. Those that can are referred to as immunogens, and this property is referred to as immunogenicity. So, not all antigens are immunogens. Obviously, since all immunogens are antigens, immunogens possess the properties of antigens. In addition to the following, the following factors contribute to the immunogenicity of an antigen: Dose: The amount of an antigen determines its immunogenicity. Very small quantities might be rapidly cleared and be unable to induce a response while very large quantities might overwhelm the system and induce a state of tolerance, depending on the nature of the antigen and on many other factors. Route: The route of administration determines availability to immune cells. Many pathogens enter the body by crossing a mucosal surface, such as the genitourinary, respiratory or gastrointestinal surfaces. Some antigens can enter through breaks in the skin. Materials introduced via the healthy digestive tract are rapidly hydrolyzed and destroyed with a reduced chance of exposure to immune cells whereas parenterally introduced materials generally remain intact and are able to induce a robust immune response. The route of administration also impacts primary type of response that is induced. 2 Microbiology & Immunology Antigen & Antibody Dr. Raymond F Adebiyi Host factors: T cells only recognize peptide antigens. These peptides are loaded into Major Histocompatibility Complex (MHC) molecules for display to T cells. While MHC molecules are encoded by a large family of alleles, a single individual will carry only a few alleles. The different MHC alleles vary in their affinity for distinct peptides. Also, there are several alleles that encode the machinery that degrade the proteins into peptides that will be loaded into MHC molecules. So, depending on the individual’s MHC and peptide processing alleles, different individuals will likely display different peptides (epitopes) from the same antigen. This variability in which peptides are generated and presented can influence the immunogenicity of an antigen. Adjuvant effects: The presence of added chemical substances (e.g. Alum) may enhance the immune response to the antigen. Adjuvants act through three basic mechanisms – by functioning prolonging the persistence of the antigen, by stimulating or modulating immune cells and by enhancing macrophage function. The portions of an antigen that bind the recognition molecules of the immune system are referred to as epitopes (or antigenic determinants). The antigen is a complex molecule and may possess several epitopes. Each epitope is a cluster of ligands held together in a mosaic. Thus, each epitope may contain amino acid sequences that are located on different parts of the polypeptide chain. Epitopes that comprise amino acids on the same chain are called linear or continuous epitopes. Epitopes formed by three-dimensional conformations (constituted by amino acids on different chains) are nonlinear or conformational epitopes. The number of epitopes on an antigen molecule is the valence. The total valence is the number of all epitopes on the molecule. However, because of the convoluted shape of the antigen, not all epitopes are readily accessible. The effective valence of the antigen in terms of its reactivity is the number of accessible epitopes. When an antigen is degraded, the conformational epitopes are the first to be lost as the molecule unfolds. Haptens are small molecules that are non-immunogenic. Haptens can be recognized by antibodies but the binding is below the threshold required for immune response. Thus, haptens possess reactivity, but because of their small size, typically only possess one epitope. Haptens may become immunogenic when they become linked to larger molecules, referred to as carrier proteins, to bring the total mass above the threshold. Haptencarrier conjugates behave like true antigens. Immunological reactivity to haptens may often be exhibited in the absence of any apparent linkage to carrier molecules. This is due to the phenomenon of autocoupling, in which haptens spontaneously bind to protein to form conjugates with immunogenic properties. 3 Microbiology & Immunology The Antigen Elimination Curve Antigen & Antibody Dr. Raymond F Adebiyi It is useful to determine how intravenously injected antigen is removed from the circulation and how this relates to the formation of antibody. Three phases of removal are easily detected. The first is the phase of equilibration. This takes place 10-30 minutes after injection and represents the time required for equilibration of the antigen with tissues and fluids. Antigen removed during this phase can be found in the liver, spleen and lung. The second phase is that of a gradual catabolic degradation and antigen removal. This proceeds over the following 4-7 days. The third stage is that of immune elimination. This is a phase of accelerated removal due to binding of newly formed antibody with antigen. Anti-gen specific antibodies are not detectable at this point as they are bound to antigen. At the end of this phase, it is possible to detect free circulating antibody. ANTIBODY An important principle in immunity is recognition – the ability of the immune system to recognize “nonself”. For the adaptive immune response (response mediated by B and T lymphocytes), a critical part of recognition is the ability to recognize a diverse array of epitopes. Antigen recognition by B lymphocytes is facilitated by the B cell receptor (BCR). Recognition of antigen through the BCR results in clonal expansion and differentiation of B cells into plasma cells. Plasma cells are essentially factories for the production of antibodies, or immunoglobulins, which are the secreted form of the BCR. Antibodies play a critical role in the adaptive immune response. This section will discuss the basic structure of antibodies, their tissue distribution and biological (or effector) functions. Generation of Antibody Diversity and Specificity Single B cells make antibodies of only one specificity and that specificity is determined by the structure of the antigen-binding site. The structure of the antigen-binding site is determined by the amino acid sequence of said antigen binding site. That amino acid sequence is generated by random gene rearrangement of immunoglobulin gene segments. Within a particular B cell, the immunoglobulin germ line genes are rearranged to include a single set out of many possible Variable (V), Diversity (D) and Joining (J) gene segments. For the  light chain, for example, 4 Microbiology & Immunology Antigen & Antibody Dr. Raymond F Adebiyi there are functional 30 V gene segments, 5 J gene segments and 1 C gene segment. For the heavy chain, there are up to 65 functional V gene segments, 27 D gene segments, 6 J gene segments and 9 C gene segments. Clearly, the rearrangement of these genes results in billions of possible combinations and this process of rearrangement accounts for antibody specificity. Rearrangement of the immunoglobulin genes occurs during the earliest stages of B cell development. Additional diversity is generated by the process of somatic hypermutation. This process results in the addition of random nucleotides into the antigen-binding region (V region) of the BCR and further modifies the avidity and affinity of antibody molecules. Somatic hypermutation occurs in during the late stage of B cell differentiation, after contact with antigen. Antibody Structure The basic structure of an antibody molecule is shown at right. The molecule is comprised of 4 polypeptide chains, 2 identical heavy chains and 2 identical light chains. The two heavy chains are linked together by disulfide bonds and each light chain is linked to a heavy chain by disulfide bonds. The antigen binding region of heavy and light chains are located at the amino-terminal of the molecule and are referred to as the variable or V domains (VH and VL, respectively). The constant domains (CH and CL) make up the constant region and define the effector function of the molecule. 5 Microbiology & Immunology Antigen & Antibody Dr. Raymond F Adebiyi While the specificity of an antibody molecule is defined by the amino acid sequence of the V region, the sequence variability is not evenly distributed across the V region. (Recall that the specificity of the antibody molecule is determined by the random gene rearrangements of the heavy and light chains occurring during B cell development) There are three regions within both the VH and VL chains that have particular variability. These hypervariable regions are separated by framework regions that provide the structural framework. When the antibody molecule assembles into its three-dimensional structure, the hypervariable regions form loops at the surface of the molecule that will interact with antigen. These loops are often termed the complementarity-determining regions (CDRs). The shape of the antibody molecule is variable, from a straight line to a ‘T’ although a Y shape is the most stable, due to steric and other factors. Studies using proteolytic enzymes (proteases) have revealed a lot about the structure of the antibody molecule and the functions of its component parts: Papain cleaves the antibody molecule into 3 fragments: 2 identical Fab (Fragment antigen binding) fragments and one Fc (Fragment crystallizable – it would readily crystallize) fragment. The Fab fragments contain the variable regions of both the heavy and light chains; they contain a complete light chain paired with the VH and CH1 domains of the heavy chain. Importantly, the Fab portion contains antigen-binding activity of the antibody molecule. The Fc fragment contains the CH2 and CH3 domains of the heavy chain and comprises the portion of the antibody molecule that determines the effector function. The term Fc reflects the properties of the domain: constant sequence, crystallizable, complement binding, complement activating, the domain with the carboxyl (COOH) terminal and cell binding via the FcR. Pepsin cuts the antibody molecule on the carboxyl-terminal side of the disulfide bonds. This cleavage produces the F(ab’)2 fragment made up of the 2 antigen-binding arms of the antibody molecule linked together. Studies using these fragments showed that the Fab fragment was unable to cross human placenta while the Fc fragment could cross the placenta. This result provided conclusive evidence that the ability of IgG to cross the placenta was an active process involving binding of the Fc domain, rather than passive diffusion of the entire molecule. So, to summarize this section on digestion of antibody molecules: Enzymatic cleavage of the IgG molecule with papain yields three fragments – two monomeric Fab fragments and one Fc fragment IgG 2Fab + Fc Digestion with pepsin yielded entirely different results. The molecule was broken into two fragments, one large dimeric fragment and a truncated Fc fragment IgG (Fab’)2 + Fc’ 6 Microbiology & Immunology Allotypes, Idiotypes and Isotypes Antigen & Antibody Dr. Raymond F Adebiyi Allotypic variations refer to genetic differences between individuals within a species involving different alleles at a given locus. Allotypic variations have no known functional significance. Allotypes occur mostly as variants of heavy chain constant regions. Allotypes are inherited as dominant Mendelian traits but have not been found in all Ig classes. Each of the four IgG subclasses has a set of allotypes (Gm allotypes, e.g. IgG3m). IgA allotypes (Am allotypes) have also been described. No allotypes have been found for the other Ig classes. Idiotypic variation refers to the variations in the variable domain, particularly in the hypervariable region (CDR). These variations determine the antigen binding specificity of a given antibody. Private idiotypes are those that are specific for individual B-cell clones. Public idiotypes are shared between different B-cell clones. Antibodies exist in different forms, or isotypes or classes. This isotypic variation is achieved through the linking of different constant region genes (located downstream of the V-genes on the heavy chain gene locus) with the same variable region. The effector function of the antibody molecule is defined by its isotype. Antibody isotypes possess different C regions but have identical Fab regions. Therefore, isotypic variation results in antibodies with the same specificity but different biological functions. 7 Microbiology & Immunology Antibody Isotypes Antigen & Antibody Dr. Raymond F Adebiyi An antibody’s heavy chain determines it’s tissue distribution and effector function. There are five heavy chain classes, Immunoglobulin (Ig)M, IgD, IgG, IgA and IgE. These classes are also called isotypes. Genes for isotypic variation generate the five classes of antibodies, which are determined by the heavy chain domains IgM ( chains), IgG ( chains), IgA ( chains), IgE ( chains) and IgD ( chains). Antibody subclasses, IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2, are based on Fc domain differences. Different isotypes of antibodies have different tissue distributions and different effector functions. Next we’ll discuss the structure, tissue distribution and effector function of each antibody isotype. IgM is the first antibody secreted in an immune response. It is the “early” antibody. The presence of IgM antibody indicates that the antigenic exposure was a recent event. IgM is secreted as a pentamer with five identical units bound by a joining “J” chain. The molecule is thus very large with 10 binding sites. Because of its large pentameric structure, IgM is found mainly in the blood and can be found in the lymph. IgM binds very strongly to pathogens with repetitive epitopes, such as polysaccharides. IgM is also a powerful activator of the complement cascade due to its size and the ready formation of immune complexes. IgG is the second antibody to be secreted, late antibody, but it is secreted in very large amounts and it has a longer half-life than IgM. IgG is therefore the most abundant antibody in blood and lymph. IgG subclasses (IgG1, IgG2, IgG3 and IgG4) possess various biological activities and greatly enhance the functional capacity of IgG. They can readily diffuse into extracellular space due to their great flexibility, IgG1 and IgG3 readily cross the human placenta, providing protection for the developing fetus. IgG antibodies can activate the classical complement pathway. They also serve an important role in opsonization, or coating pathogens and marking them for phagocytosis by phagocytic cells expressing Fc receptors. NK cells express an Fc receptor, CD16/FcRIII, which is specific for IgG1 and IgG3. CD16 engagement results in antibody dependent cell-mediated cytotoxicity, ADCC. IgA occurs as a monomer and as a dimer. Trimeric and tetrameric forms of IgA have also been described. Monomeric IgA is secreted into the bloodstream. Dimeric IgA contains a J chain and a secretory component (SC). It is transported across the epithelium and is found in the lumen of the gut as well as in secretions (saliva, tears, milk) and on mucus membranes (respiratory and intestinal). More IgA is produced in the gut than all other Ig from other sources combined. As IgA is predominately found along mucosal surfaces where complement and phagocytes are not present, the main function of IgA antibodies is to neutralize pathogens. IgE binds to high affinity receptors (FcR) on mast cells (which are found beneath the skin and mucosa, and along blood vessels in connective tissue) and basophils. As such, is found at very low levels in the blood and extracellular fluid. Bound to these FcE receptors, lgE is highly efficient at triggering mast cells to release their granule contents and trigger the elimination of certain pathogens, namely helminthic parasites. IgE also plays a major role in the pathogenesis of allergic reactions (Type I hypersensitivity). IgD is the least understood of the immunoglobulins. Secreted IgD has no known function. IgD is however expressed on the surface of B cells, and, with IgM, acts as the B cell receptor (BCR) for antigen. 8 Microbiology & Immunology Antigen & Antibody Dr. Raymond F Adebiyi It is important to understand that antigen-inexperienced B cells only express surface-bound antibodies that are of the IgM and IgD isotypes. Only after a naïve B cell encounters and receives help from helper T cells (specifically, activated CD4 T cells) can B cells switch their heavy chain isotype to IgG, IgA, or IgE. Help from T cells comes in the form of cytokines and the isotype to which the B cell will switch depends on the cytokine secreted by the T cell (see below). Fc Receptors Fc receptors are specialized receptors expressed by immune cells. They bind to the Fc portions of antibodies and trigger a variety of effector functions. Different Fc receptors bind different Fc regions on the various antibody isotypes. When antibodies bind to multimeric antigens or multimeric antigenic particles, they aggregate; Fc receptors can bind the Fc portions of the antibodies in these aggregates and this triggers the cell to initiate effector functions like phagocytosis or ADCC. In the case of Fc receptor (which is expressed by mast cells, eosinophils and basophils), IgE tends to stay bound to the Fc receptor, and when the specific antigen comes along, those Fc receptor-bound IgEs will aggregate on the cell surface and trigger release of granules. 9 Microbiology & Immunology Antigen & Antibody Dr. Raymond F Adebiyi Helper T cells regulate isotype switching The cytokines produced by an activated CD4 T cell can induce activated B cells to switch to a particular isotype. The activated helper T cell (which was influenced by the cytokines produced by the APC) now influences the activated B cell – all in order to make the antibody best suited for elimination of the pathogen! Antigen-Antibody reactions Antigen-antibody reactions are highly reversible because weak non-covalent forces hold the reactants together: Ag + Ab  (AgAb) The determinants of reaction equilibrium include the relative concentrations of the reactants, reaction temperature and pH and the binding affinity of the reactants. In general, the biological outcomes of antigen-antibody reactions depend on the nature of the antigen. Agglutination – whole cells or aggregated antigens Precipitation – soluble antigens Flocculation – particulate antigens Neutralization – toxins or live organisms 10