IAPTER 1 Basic Immunology

&IAPTER Basic Immunology The Immune Response A knowledge of immunology is essential in developing ELISAs Information about the specific system and similar systems already studied from the biochemical and immunological aspects is important to allow development of assays and to determine the significance of results It is not the intent of this chapter to provide an in-depth understanding of immunology; however, it is necessary for the ELISA operator to have a basic understanding of: 1, The immunology of infectious diseases The propertiesof certain componentsof the immune system Aspects of serology Mammals possess a system of surveillance called the immune system that protects them from disease-causing (pathogenic) microorganisms, such as viruses, bacteria, and parasites The immune system specifically recognizes and eliminates pathogens The protection afforded by the immune system of the mammal is divided into two functional divisions, namely, the innate immune system and the adaptive immune system, both of which respond specifically to these foreign substances Innate immunity acts as the first line of defense against infectious agents, and most potential pathogens are checked before they can establish infection If these defenses are overcome, then the adaptive immune system is activated The adaptive system produces a specific reaction against each infectious agent, and also remembers that particular agent and can prevent it causing disease in the future Most of the applications of ELISA involve studies on the adaptive immune system, so this will be featured in more detail Excellent books on basic immunology have been written, in particular (I), which has extensive references for specific areas outlined below Basic Immunology 1.1 Innate Immunity The factors involved in innate immunity include biochemical and physical barriers, e.g., the skin, acting as an impenetrable barrier to infectious agents, and the presence of lysozyme in tears, which destroys bacteria, such as S aureus The key difference in this system from that of the adaptive immunity is that resistance is not improved by repeated infection and that the system is aspecific in nature Thus, if organisms penetrate an epithelial surface, they encounter phagocytic cells of the reticuloendothelial system (RE), where the cells are of many types derived from bone marrow cells Their function is to engulf, internalize, and destroy infectious agents For this purpose, they are placed strategically where they might encounter particles, e.g., the Kupffer cells of the liver line the sinusoids along which blood flows The blood phagocytes include neutrophil polymorph and the blood monocytes, both of which can migrate into tissues as a result of an invasive stimulus Other cells, such as natural killer (NK) cells, are leukocytes capable of recognizing cell surface changes on virus-infected cells Such cells bind and kill cells under the influence of substancescalled interferons, which are produced by the virus-infected cells or sometimes by lymphocytes Other factors involved in innate immunity involve certain serum proteins These are referred to as acute-phaseproteins The concentration of such proteins rises dramatically on and is maintained throughout infection The various proteins have defined properties and produce protective effects through complex interactions with other serum components, such as complement followed by lysis of disease agents Visible signs of an early immune response are observed in inflammation, which is the body’s reaction to an injury, such as invasion with an infectious agent Three major events occur, namely: 1, An increasedblood supply to the infected area, An increasem the permeability of the capillarrescausedby retraction of endothelial cells, allowing larger moleculesto crossthe endothelium,e.g., soluble mediators Migration of leukocytes( neutrophils,polymorphs,and macrophages)from capillaries to surroundmgtissues, These features, whereby phagocytes are attracted to sites of injury, are important in immunity and initiate all levels of potential innate immune mechanisms Antigens 1.2 Adaptive Immunity Innate immunity relies on stimulation of factors through aspecific recognition of infectious agents Problems arise when the recognition process is not activated, e.g., when phagocytes are unable to recognize the infectious agent either because they lack a suitable receptor for the agent or because the agent does not activate soluble factors What is needed in such a situation is a specific molecule that can attach at one end to the infectious agent and at the other end to the phagocytic cell Such molecules, called antibodies, are produced in the mammalian systems Antibodies are produced by B lymphocytes of the adaptive immune system, which act as flexible adaptors between infectious agents and phagocytes Any particular antibody molecule can bind only to one type of infectious agent, and the other end of the molecule binds to the phagocyte by way of a receptor, the Fc receptor A stylized structure of an antibody is shown in Fig 1A Figure 1B shows that IgG is a rather bulky structure when examined at the molecular level Antibodies are effectively bifunctional molecules One part, which is extremely variable between different antibodies, binds to all the various infectious agents, whereas the second, constant portion binds to receptors of cells and also activates complement Antigens The mammalian immune system has the ability and capacity to recognize surface features (topography) of foreign macromolecules or microorganisms that are not normal constituents of that mammal (e.g., pathogenic microorganisms) This recognition of surface features is normally specific, and the components of the mammal that carry out this specific recognition of surface features of macromolecules or microorganisms are protein molecules called antibodies Foreign substanceshave specific surface features, called antigens, that antibodies recognize The portion of the antigen to which the antibody binds is called the antigenic determinant or epitope Antibody specifically binds to epitopes on the antigen by multiple noncovalent interactions similar to the interactions that confer specificity to enzyme-substrate reactions The part of the antibody that binds to the antigenic determinant is termed the antigen-combining site or paratope (which is complimentary to the epitope) An antigen eliciting a response from the immune system is referred to as an immunogen Microorganisms, macromolecules, such as foreign Basic Immunology A DISUPHIDE NH+ BON6 LIGHT HEAVY B CHAIN CHAIN Heayy chain Fig (A) Representationof basic structureof an IgG molecule (B) Model of IgG molecule basedon X-ray crystallographicanalysis proteins, nucleic acids, carbohydrates, polysaccharides, and so forth, are usually effective immunogens Molecules with mol wt below 5000 usually are not effective immunogens However, many of these small nonimmunogenic molecules, when covalently attached to a large molecule, can stimulate an immune response These molecules, which are nonimmunogenic, are termed haptens, and the large molecules to which they can be covalently attached, generally proteins, are termed carriers Once the hapten is attached covalently to the carrier protein and introduced into an organism, a specific antibody response to the hapten and the carrier (if the latter is recognized) occurs This antibody response can be specific to the hapten Thus, nonimmunogenic molecules (haptens) can be recognized by an organism when covalently attachedto a carrier protein Antigens 2.1 Antigen Presentation, Processing, and Recognition Lymphocytes account for between 20 and 80% nucleated cells in the blood, and over 99% nucleated cells in lymphatic fluid Lymphocytes contact and respond to antigens in specialized lymphoid organs Included in these specialized organs are the spleen, thymus, lymphatic tree and the lymph nodes positioned along them, bone marrow, and Peyer’s patches (appendix, adenoids, tonsils [Bursa of Fabricius]) The lymphoid system has three principal functions, namely: Concentration of antigens from all parts of the body into lymphoid organs Circulation of lymphoid cells through these organs to ensure antigen exposure to antigen-specific lymphocytes in a short period of time Transmission and dissemination of the products of the immune response, e.g., antigen-specific effector B + T-cells, humoral antibodies, throughout the body Antigens are collected and processed by different lymphoid organs depending on their route of entry into the body, i.e., respiratory system, gastrointestinal tract, skin, vector transmission, venereal, and so on Antigen processing in all these lymphoid organs involves macrophages,a short period of time after infection (injection) The antigen(s) becomes incorporated in special vesicles (phagolysosomes) within the macrophage The macrophage cell surface either retains or receives a small amount of immunogenic material for presentation to antigen-specific lymphocytes Binding of this macrophage surface antigen to a B-cell or T-cell (presentation of antigen) induces a general activation of the cell This process, known as blast-transformation, causes the B-cell (or T-cell) with the appropriate receptor specificities to recognize the antigen-presenting macrophage to enlarge Such activated cells initiate DNA synthesis, divide, and give rise to effector cells and memory cells of the B-cell (or T-cell) lineage Most memory B-cells re-enter the general circulation, whereas most effector B-cells are retained in the lymph node An individually activated B-cell proliferates and differentiates to form plasma cells that begin to produce identical antibodies with a single antigen specificity (at the rate of 3000-30,000 molecules/cell/s) An organism’s total response even to a simple antigen is almost always heterogeneous with respect to antibody specificity because most antigens have multiple epitopes, which can trigger the activation of different Basic Immunology B-cells Consequently, the serum of the host reflects the heterogeneous collection of immunoglobulin molecules previously secreted Although individual B-cells are committed to produce ,one or at most two antibody isotypes, the B-cell response to any antigen can produce antibody molecules of all five classes of immunoglobulins Since antibody molecules of different classes differ in their heavy-chain-constant regions (Fc), they may exhibit identical antigen-binding specificities and, hence, identical variable regions Antibodies of the five classes mediate different physiological effector functions, and are present in serum at different concentrations and for different half-life periods The different classes are also produced in different relative amounts in primary and secondary immune responses 2.1.1 IgM This is the first antibody produced in responseto an irnmunogen and is particularly effective against invading microorganisms It is a pentamer in serum, and although the affinity of each active site on the pentamer (10 in number) for an epitope may be low, the overall avidity of the pentamer for a complex antigen is high becauseof the repeatingnature of epitopes on many cell membrane antigens.Becauseit is presentas a pentamer in serum, IgM is about 1000 times more effective (on a molar basis) at agglutinating cells by crosslinking them, than a monomeric antibody against the same epitope IgM coated onto the antigen of target cells stimulates target cell ingestion by macrophagesand target cell destruction (lysis) by compliment fixation 2.1.2 IgG This monomeric antibody is normally produced later in the immune response than IgM This is the most prevalent antibody in blood and tissue spaces, and is capable of fixing complement It also activates macrophage ingestion of opsonized (coated) antigen particles IgG is the only class of antibody that can cross the placenta to provide passive immunity to the developing fetus Normally, the affinity of IgG antibodies toward a specific antigen increases with time after immunization, a process known as affinity maturation 2.1.3 IgA This is also produced later in the immune response than IgM It can exist as a monomer, dimer, or trimer of the basic Y-shaped structural unit IgA antibodies are important at numerous epithelial surfaces and Adaptive Immunity and Clonal Selection act as a potential protective barrier at several points of entry, e.g., gastrointestinal tract, respiratory tract, genitourinary tract, eyes, and so forth Some epithelial cells produce a polypeptide, called the secretory component, which complexes to the Fc region of IgA and mediates their transport across the epithelial cell surface to the lumen IgA B-cell precursors are especially frequent in lymphoid organs draining the gastrointestinal tract and in the mammary glands IgA is the major immunoglobulin in colostrum and milk, and is also present in sweat, tears, and saliva 2.1.4 IgE This monomeric antibody is heat-labile It is present in blood in very low concentrations IgE antibodies are produced in response to infection mainly the helminth parasites and in allergic atopic conditions IgE antibodies can bind via their Fc regions to mast cells or blood basophils Further interaction of this bound IgE with a cognate (known and recognized by IgE) antigen can trigger cell degranulation and the liberation of vasoactive compounds, such as histamine and heparin 2.1.5 IgD This monomeric antibody is present only in minute concentrations in blood Its functions are unknown Adaptive Immunity and Clonal Selection The immune system as a whole can specifically recognize many thousands of antigens The specificity of the adaptive immune response is based on the specificity of the antibodies and lymphocytes, and since it has been shown that each lymphocyte is only capable of recognizing one particular antigen, this means that the lymphocytes recognizing any particular antigen are a very small proportion of the total Thus, we have to explain how an adequate response to an infectious agent is mounted The answer is clonal selection, whereby antigen binds to a small number of cells that can recognize it and induces them to proliferate Thus, the antigen selects the specific clones of antigen-binding cells This is illustrated in Fig This process occurs in both B-lymphocytes, where they mature into antibody-producing cells, and T-lymphocytes, which areinvolved in the recognition and destruction of infected cells A basic requirement for the production of an antibody response is that the immunogen possessessurface features that are recognized as foreign in the animal into which it is introduced or in which it occurs Basic Immunology Antigen selection Production of antibody Fig The antibody-producing cells (B-cells) are programmed to make a single antibody only The antibody is placed on an Fc receptor on the cell’s surface Each B-cell has a different receptor, and antigen binds to those cells with the appropriate receptor The cells become stimulated to multiply and mature onto antibody-producing cells and memory cells, which can live longer All the cells have the same antigen-binding capacity Antibodies Antibodies are fundamental reagents in ELISA, and the determination of their presence and/or concentration in the blood is vital in understanding disease processes and in diagnosis of disease A knowledge of the properties of antibodies is fundamental to the development of specific assays An understanding of the variation in antibody composition of different mammals is also important Antibodies ANTIGEN BINDING (PARATOPE) SITE CARBOHYDRATE CARBOHYDRATE- FC I@ s s c#& HEAVY WSULPHJDE BOND s s s s CHAIN I I COO- coo- Fig Structural elements of an IgG molecule 4.1 Antibody Structure and Function Antibodies form a group of glycoproteins present in the serum and tissue fluids of all mammals The group is also termed immunoglobulins, indicating their role in adaptive immunity All antibodies are immunoglobulins, but not all immunoglobulins are antibodies, i.e., not all the immunoglobulin produced by a mammal has antibody activity Five distinct classes of immunoglobulin molecule have been recognized in most higher mammals These are immunoglobulin (Ig) G (IgG), IgA, IgM, IgD, and IgE These classes differ from each other in size, charge, amino acid composition, and carbohydrate content There are also significant differences (heterogeneity) within each class The basic polypeptide structure of the immunoglobulin molecule is shown in Fig 10 Basic Immunology The basic structure of all immunoglobulin molecules is a unit of two identical light (L) polypeptide chains and two identical heavy (H) polypeptide chains linked together by disulfide bonds The class and subclass of an immunoglobulin molecule are determined by its heavy-chain type Thus, in the human, there are four IgG subclasses, IgGl, IgG2, IgG3, and IgG4, which have heavy-chains called 1,2,3, and The differences between the various subclasses within an individual immunoglobulin class are less than the differences between the different classes.Thus, IgGl is more closely related to IgG2, and so on, than to IgA, IgM, IgD, or IgE The most common class of immunoglobulin is IgG IgG molecules are made up of two identical light chains of mol wt 23,000 Daltons and two identical heavy chains of mol wt 53,000 Daltons Each light chain is linked to a heavy-chain by noncovalent association, and also by one covalent disulfide bridge For IgG, each light-heavychain pair is linked to the other by disulfide bridges between the heavy chains This molecule is representedschematically in the form of a Y, with the amino (N-) termini of the chains at the top of the Y and the carboxyl (C) termini of the two heavy chains at the bottom of the Y-shape A dimer of these light-heavy-chain pairs is the basic subunit of the other immunoglobulin isotypes The structures of these other classes and subclasses differ in the positions and number of disulfide bridges between the heavy chains, and in the number of light-heavy-chain pairs in the molecule IgG, IgE, and IgD arecomposed of one light-heavy-chain pair IgA may have one, two, or three light-heavy-chain pairs IgM (serum) has five light-heavy-chain pairs, whereas membrane-bound IgM has one light-heavy-chain pair In the polymeric forms of IgA and IgM, the lightheavy-chain pairs are held together by disulfide bridges through a polypeptide known as the J chain In both heavy and light chains, at the N-terminal portion, the sequences vary greatly from polypeptide to polypeptide In contrast, in the C-terminal portion of both heavy and light chains, the sequences are identical Hence, these two segments of the molecule are designated variable and constant regions For the light chain, the variable region (V) is approx 110 amino acid residues in length, and the constant region (C) of the light chain is similarly about 110 amino acids in length The variable region of the heavy chain (Vu) is also about 110 amino acid residues in length, but the constant region of the heavy-chain (C,) is about 330 amino acid residues in length Basic Immunology 20 be capable of neutralizing the effect of or destroying the inciting agents Such antibodies are known as protective antibodies, and their complementary antigens are called protective antigens The protection conferred by active immunization is not immediate, as in passive immunization, because the immune system requires a length of time in order to process such antigens and produce protective antibodies However, the advantage of active immunization is that it can be long-lasting, and restimulation by the same antigens present in pathogens leads to an anamnestic response It is important to recognize that the immunity produced to pathogens, following active immunization, is only as broad as the antigenie spectrum of the preparation used for immunization Protection can be afforded using different approaches in the formulation of vaccines 4.3.3 Live Vaccines These may be attenuated by passageof agent, e.g., viruses, in unusual hosts so that they become nonpathogenic to animals that are vaccinated Usually these are good vaccines, since they supply the same antigenic stimulus as disease agent There can be problems of reversion to pathogenic agent Some replication of the agent usually occurs 4.3.4 Modified Vaccines-Whole-Disease Agent These can be grown and then chemically modified, e.g., heat-killed, nucleic acid-modified (mutagens), formaldehyde-treated These are potentially good vaccines, in that a full antigenic spectrum is given The antigenic mass has to be high, since there is no replication to challenge the immune system Repeat vaccinations are common to elevate antibody levels 4.3.5 Purified Antigens “Protective” antigens can be identified and used as protein, polypeptide, and peptide immunogens, usually with adjuvants These vaccines are usually not as good as those in which the total antigenic spectrum is used They have the advantages of being able to synthesize products on a large scale by chemical methods, e.g., as peptides, and are noninfectious 4.3.6 DNA Technology Products Genes producing particular immunogens can be inserted into replicating agents, so that their products are expressed This is a novel approach, e.g., in vaccinia, where more than one gene can be inserted 21 Antibodies 4.3.7 Generalities Most vaccines are administered by either subcutaneous or intrarnuscular injections When vaccinating large herds of animals, other techniques, such as high-pressure jet injections, may be employed It is obvious that the risk of administering unwanted/contaminating organisms/antigens should be minimal Hence, sterile administration of vaccines is indicated Subcutaneous or intramuscular vaccination should induce all antibody isotypes given the fact that the inciting antigens are capable of doing so Hence, the immunoassayist must consider whether total antibody assays, isotype-specific assays, or assays to detect antigen clearance are to be utilized to assessthe effects of vaccination Some antigens may be administered orally, e.g., poliomyelitis vaccines in humans, by incorporation in food or drinking water, e.g., in poultry flocks, or by inhalant exposure of aerosolized vaccine, e.g., diseases of respiratory tract In these instances, the production of local antibodies to prevent the ingress of pathogens through the gastrointestinal or respiratory tree barriers is sought In such situations, the immunoassayist must decide whether an assay for isotype-specific antibodies, notably IgA, may provide a deeper insight into the benefits of vaccination than an assay for total antibody In some instances, where an infectious disease agent is endemic and vaccination, especially of newborns, is indicated, it may prove difficult or impossible to differentiate the beneficial effects of vaccination because residual levels of antibody may be present in nonvaccinated stock Such factors must be borne in mind !when assays are developed to determine the immunological status of large groups of mammals 4.4 Antibody Production in Response ‘to Infectious Agents It is not the function of this chapter to catalog the humoral immune responses produced in mammals in response to the variety of infectious disease agents, such as viruses, bacteria, fungi, protozoa, helminths, and arthropods Such information may be obtained from textbooks and specialized review articles This section will deal with the general considerations of the host-parasite relationships, with specific reference to the production of antibodies to pathogens As already mentioned, most infectious diseases are transmitted by aerosolization, close contact, or vectors, and their final location may be 22 Basic Immunology distant from their point of deposition Therefore, these pathogens can have multiorgan involvement Similarly, many pathogens, but not all, have the capacity to divide within the mammalian body, and in such instances, the numbers and amounts of antigens produced will increase over time and will be proportional to the number of pathogens at the time of sampling Where pathogens not divide or reproduce in the mammalian host, the amount of antigens produced may be directly proportional to the infective dose Hence, in devising assays for infectious disease agents, the immunoassayist must take into account whether high concentrations of antigens and/or antibodies, or low concentrations of antigens and/or antibodies are to be sought Where antibody titers of c 150 are anticipated, serum dilutions of cl:50 or possibly c 1:10 for the test serum must be employed Previous knowledge of specific host-parasite systems will prove invaluable in devising more specific and sensitive enzyme immunoassays Although many nonimmunological mechanisms exist for the removal of pathogens from the body (e.g., lysozyme, iron-binding proteins, myeloperoxidase, lactoperoxidase, compliment, basic peptides and proteins, and so on), it is generally recognized that the immune system plays a vital role in the control and destruction of pathogens For this reason, the measurement of antibody or antigen by sensitive assays, such as ELISA, provides a useful indicator for the assessment of immune status When an infectious agent enters the mammalian body, the first components recognized as foreign are surface components of that pathogen This host-pathogen interface plays a vital role in the control of infectious diseases, not only in its involvement in stimulating the early humoral immune response, but also in its involvement in mediating protective immune responses.Immune responsesthat reduce pathogen numbers by lysis, agglutination and/or phagocytosis, or reduce the antigen load, and so forth, are normally regarded as protective responses, and such antibodies directed against specific epitopes on the pathogens can be sought by the immunoassayist in an effort to correlate protective responses with clinical betterment However, insight into the molecular basis of such interactions is necessary before immunoassays can be developed to demonstrate protective responses (e.g., knowledge of the immunochemistry of the surface-exposed molecules and their epitopes, knowledge of specific antibody isotypes that mediate these responses) Since infectious disease agents stimulate antibody production, these 23 Antibodies antibodies can prove useful to the immunoassayist for detecting exposure to pathogens We have seen that when the antigens of pathogens are recognized by the host, an antibody response ensues, initially of the IgM isotype and followed by the IgG isotype, together with an increase in antibody affinity over time Where a variety of antibody isotypes are produced in response to infection, use can be made of this isotypic variation in order to determine the chronicity of the infection, since IgM antibody isotypes normally appear before IgG antibody isotypes Similarly, increasing levels of antibodies can indicate current infections or exacerbations of infections, whereas decreasing antibody titers can indicate past infections or successful control of current infection, In the absence of detectable free circulating antibody, either free antigen or circulating immune complexes can be detected by ELISA When antibodies specific to antigens of a pathogen are used to detect the presence of free antigen in the test sample (see trapping/capture assays), a direct correlation can be made between ELISA positivity and current infection, Where protective mechanisms occur, destruction of the pathogen is the outcome This is accompanied by the release of previously internal components, which, if antigenic, will stimulate the production of specific antibodies Thus, the destruction of pathogens will lead to the production of antibodies against the antigen repertoire, both surface-exposed and internal, of that pathogen Owing to the commonness of some internal antigens (e.g., enzymes, and so on), the consensus of opinion indicates that the more specific antigens of pathogens (excluding endotoxins) are surface-expressed at one time or another during development The surface-exposed antigen mosaic is normally less complex than the internal antigen mosaic of pathogens 4.4.1 Effect ofAntibody in Viral Infection Viruses as a group must enter a cell to proliferate, since they lack the biochemical machinery to manufacture proteins and metabolize sugars Some viruses also lack the enzymes required for nucleic acid replication The number of genescarried by viruses varies from to about 250, and it is worth noting how small this is compared to the smallest bacterium The illnesses caused by viruses are varied, and include acute, recurrent, latent (dormant but can recur), and subclinical The immune response ranges from apparently nonexistent to lifelong immunity, The acute infection is probably most encountered by the immunoassayist interested 24 Basic Immunology in animal diseases, but it must be borne in mind that the total knowledge of a specific disease is needed in order to devise assays relevant to specific problems Since the outer surfaces (capsids) of viruses contain antigens, it is against these antigens and the envelope that the antiviral antibodies are mounted The first line of defense (excluding interferon) is either IgM and IgG antibodies, where viruses are present in plasma and tissue fluids (vector transmitted), or secretory IgA antibodies, where viruses are present on epithelial surfaces (airborne, close contact) Some viruses that replicate entirely on epithelial surfaces (e.g respiratory tree, gastrointestinal tract, genitourinary tract) and not have a viremic phase will be controlled by secretory IgA Antibodies may destroy extracellular viruses, prevent virus infection of cells by blocking their attachment to cell receptors, or destroy virus-infected cells 4.4.2 Effect of Antibody in Bacterial Infection The role of antibody in combating bacterial infection is diverse Antibody to bacterial surface antigens (fimbriae, lipotechoic acid, and some capsules) prevents the attachment of the bacterium to the host cell membrane by blocking receptor sites Antibody can neutralize bacterial exotoxins (possibly by blocking the interaction between the exotoxin and the receptor site) Normally, IgG antibodies are responsible for toxin neutralization Antibody to capsular antigens can neutralize the antiphagocytic properties of the capsule, or in organisms lacking a capsule, antibodies to somatic antigens may serve a similar function IgG anti[...]... affect the degree and speed by which IgG replaces IgM These considerations are vital for the immunoassayist concerned with diagnosing infectious diseasesof mammals, and great care and planning 14 Basic Immunology 0 5 10 15 20 25 30 Days after primary 35 40 45 50 55 dose of antigen Fig 5 Anamnestic response following second administration of antigen Primary response following initial antigen dose has a lag... fit) Basic Immunology 16 Good tit High attraction Low repulsion High affinity Fig 7 A good fit betweenantrgenicsitesand antibody-combining sitescreates an environment for the intermolecular attractive forces to be createdand limits the changesof repulsive forces.The strengthof the single antigen-antibody bond is the affinity that reflects the summationof the attractive andrepulsive forces 4.2 .1 Affinity... or “Fab.” Each Fab consists of the variable and constant regions of the light chain and the variable and part of the constant (ChI domain) regions of the heavy-chain Therefore, each Fab carries Basic Immunology 12 ANTIGEN COMBINING SITE Heavy chains MONOVALEW FC’ MONOVALENT FE Fig 4 Enzymic cleavageof humanIgG Pepsincleavestheheavychainto give F(Ab’), andpFc’ fragments.Furtheractionresultsin greaterfragmentationof... and, therefore, the avidity equals affinity, Thus, the population reacts identically to any individual molecule in that population After diluting the monoclonal population, there is no alter- Basic Immunology 18 Specific reaction AgX Homologous Antigen Cross-reaction No reaction AgZ Determinant shared B No determinants shared Fig 8 Specificity, crossreactivity, and nonreactivity Antisera contain populations... take into account whether high concentrations of antigens and/or antibodies, or low concentrations of antigens and/or antibodies are to be sought Where antibody titers of c 15 0 are anticipated, serum dilutions of cl:50 or possibly c 1: 10 for the test serum must be employed Previous knowledge of specific host-parasite systems will prove invaluable in devising more specific and sensitive enzyme immunoassays... disease agents, might be involved in crossreactions and develop the assay accordingly, bearing in mind the type of problems associated with such crossreactions Reference 1 Roitt, I M., Brostoff, St Louis, MO J., and Male, D K (19 93) Immunology, 3rd ed., Mosby, ... sites may also contribute to the crossreaction Where all the antibodies show no recognition of the antigens available, no reaction will be seen It is important to understand the Antibodies 19 concepts of variability in (1) isotype production, and (2) affinity and affinity maturation when developing immunoassays for infectious disease agents that are normally more chronic than acute in duration Because... infectious disease agents, once contracted Re-exposure to such agents, following active immunization, will result in an anamnestic immune response, whereby the antibodies or effector cells produced will Basic Immunology 20 be capable of neutralizing the effect of or destroying the inciting agents Such antibodies are known as protective antibodies, and their complementary antigens are called protective antigens... reference to the production of antibodies to pathogens As already mentioned, most infectious diseases are transmitted by aerosolization, close contact, or vectors, and their final location may be 22 Basic Immunology distant from their point of deposition Therefore, these pathogens can have multiorgan involvement Similarly, many pathogens, but not all, have the capacity to divide within the mammalian...Antibodies 11 The N-terminal portions of both heavy- and light-chain pairs comprise the antigen-combining (binding) sites in an immunoglobulin molecule The heterogeneityin the amino acid sequencespresentwithin the