The Elisa guidebook
.pdfthe constant (ChI domain) regions of the heavy chain. Therefore, each Fab carries one antigen binding site. The third fragment, consisting of the remainder of the constant regions of the heavy chains, is readily crystallizable and is called fragment crystallizable or Fc.
Pepsin digestion cleaves the Fc from the molecule but leaves the disulfide bridge between the Fab regions. This molecule contains both antigen-combining sites and is bivalent.
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5.1.2¡ª
Antibody Classes
The five immunological classes (isotypes) can be distinguished structurally by differences in their heavy chain constant regions (i.e., mainly the Fc portion). These heavy chain classes define the corresponding Ig classes IgA, IgG, IgD, IgE, and IgM. Some classes can be divided further into subclasses.
In addition, two major types of light chains exist, based on the differences in the constant region Cl and are known as kappa (κ) and lambda (λ). Igs from various mammals appear to conform to this format. However, the subclass designation and variety may not be the same in all species examined; for example, mice have IgG1, IgG2a, IgG2b, IgG3, and cows have IgG1 and IgG2.
5.2¡ª
Antibody Production in Response to Antigenic Stimulus
The antibodies produced in a humoral response to antigenic stimulus are heterogeneous in specificity and may include all Ig classes. This heterogeneous response is owing to the fact that most antigens have multiple antigenic determinants that trigger off the activation of different B-cells. Therefore, the serum of any mammal (vertebrate) contains a heterogeneous mixture of Ig molecules. The specificities of these Ig molecules will reflect the organism's past antigenic exposure and history.
The first antibody produced in response to a primary exposure of an immunogen is IgM. When the immunogen is persistent or the host (mammal) is reexposed to the immunogen, other classes of antibody may be produced as well as IgM. The body compartment in which the immunogen is presented can determine the predominant antibody isotype produced (e.g., IgA in the gastrointestinal [GI] tract). In general, primary exposure to an immunogen stimulates the production of IgM initially, followed by the appearance of IgG), as shown in Fig. 7.
If no further exposure occurs, or the immunogen is removed by the mammal, a low level of IgM and IgG can be detected. If reexposure occurs, a similar peak of IgM antibody is produced that declines in a similar kinetic manner to the primary IgM response, but the IgG response is not only more rapid (over time) but also reaches higher serum levels that persist for a longer period of time. This IgG response to reexposure is known as the anamnestic response. This is illustrated in Fig. 7.
In cases in which complex antigens occur, as in infectious diseases, the dosage (infection level), type of antigen (viral, bacterial, protozoan, helminthic), route of infection (oral, respiratory, cutaneous), and species of mammal infected (cow, pig, camel, human) will all affect the degree and speed by which IgG replaces IgM.
These considerations are vital for the immunoassayist who is concerned with diagnosing infectious diseases of mammals, and great care and planning should
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Fig. 7.
Anamnestic response following second administration of antigen. Primary response following initial antigen dose has a lag phase in which no antibody is detected (4¨C5 d). This is followed by a lag phase in which antibody is produced. A plateau phase follows in which antibody titers stabilize, after which a decline in titer is observed. On secondary stimulation, there is an almost immediate rise in titer and higher levels of antibodies are achieved that are mainly IgG.
be exercised before undertaking such immunoassays. Note also that at this stage, different infectious disease agents can stimulate different antibody isotypes. For example, certain viral pathogens stimulate predominantly IgM agglutinating responses, bacterial polysaccharides stimulate IgM (and IgG2 in humans) antibodies, and helminthic infections stimulate the synthesis of IgE antibody.
In general, it can be stated that during the development of immunity to infectious disease agents, the antibodies produced become capable of recognizing antigens better, as demonstrated by improved antigen-antibody interaction. The multispecificity of antibody molecules (i.e., the ability to combine with a variety of epitopes containing similar molecular structures) is dependent not only on the heterogeneity of the epitope in question, but also on the molecular construction of the antigen-reactive sites (paratopes) of the antibody molecules.
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Fig. 8.
A good fit between antigenic sites and antibody-combining sites creates as environment for the intermolecular attractive forces to be created and limits the chances of repulsive forces. The strength of the single antigen/antibody bond is the affinity that reflects the summation of the attractive and repulsive forces.
5.3¡ª
Affinity and Avidity
The binding energy between an antibody molecule and an antigen determinant is termed affinity. Thus, antibodies with paratopes that recognize epitopes perfectly will have high affinity (good fit) for the antigen in question, whereas antibodies with paratopes that recognize epitopes imperfectly will have low affinity (poor fit). Low-affinity antibodies in which the fit to antigen is less than perfect will have fewer noncovalent bonds established between the complex, and the strength of binding will be less, as shown in Fig. 8.
With simple immunogens containing few epitopes, as the antibody response develops (in response) to this immunogen, its recognition by antibody will become better or closer, that is, low-affinity antibodies will be replaced by high-affinity antibodies, which will cause the interaction between antigen and antibody to be more stable. Antibodies produced later during infection are generally of higher affinity than those produced early on during infection. Hence, the IgG antibodies produced in response to reexposure will be of higher affinity than those produced in response to initial exposure.
In a serum sample in which there has been polyclonal stimulation of antibody production by antigen, a variety of affinities will be present within the
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antibodies. The match (fit) between antibodies to that antigen will be variable, and the antibodies present in that serum sample will bind to antigen differentially. Thus, not only can an antigen stimulate different antibody isotypes but also antibodies with different affinities for the antigenic determinant. Avidity can be regarded as the sum of all the different affinities between the heterogeneous antibodies contained in a serum and the various antigenic sites (epitopes). It is important to realize that the avidity of a serum may change on dilution because an operator may be diluting out certain populations of antibodies.
As an example, we could have a serum containing a low quantity of antibodies showing high affinity for a particular complex antigen and a high quantity of low-affinity antibody. Under immunoassay conditions in which that serum is not diluted greatly, we would have competition for antigenic sites between the highand low-affinity antibodies, and the high-affinity antibodies would react preferentially. On dilution, however, the concentration of the high-affinity antibodies would be reduced until we would be left only with low-affinity antibodies. Such problems are important when an operator is using immunoassays to compare antigens by their differential activity with different antisera. The dilution of any serum can affect its ability to discriminate between antigens owing to the dynamics of the heterogeneous antibody population (relative concentrations and affinities of individual antibody molecules).
Such problems of quality and quantity do not apply to mAbs, because, by definition, the Ig molecules in the population are identical. They all have the same affinity and therefore the avidity equals affinity. Thus, the population reacts identically to any individual molecule in that population. On diluting the monoclonal population, there is no alteration in the affinity/avidity of the serum, and a change noted for reaction between the mAb and antigen must be from changes on the particular antigen.
Figure 9 illustrates crossreactions between sera and different antigens. Here, specific reactions occur in which all the antibodies have ''best fit." When two antigens share a similar antigen, crossreactions will be observed. The two non-identical sites may also contribute to the crossreaction. When all the antibodies show no recognition of the antigens available, no reaction will be seen. It is important to understand the 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.
As antigens are introduced into different compartments of the mammalian body they can stimulate the production of different antibody isotypes. Local antibody responses in the GI tract and the respiratory tree are predominantly IgA isotypes, whereas those in the other major compartments are predominantly IgG (IgM). Certain sites in the body (e.g., testes) are immunologically privileged and stimulate lower antibody responses to immunogens. Most
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Fig. 9.
Specificity, crossreactivity, and nonreactivity. Antisera contain populations of antibodies. Each population is directed against a different determinant (A, B, and C). Antigen X and Y share a determinant (B); thus, antiserum against X will react withantigen Y (crossreact) as well as reacting specifically with antigen X. Antiserum against antigen X does not react with antigen Z since no determinants are shared.
infectious diseases are transmitted by aerosolization, close contact, or vectors; thus, their route of transmission is variable. In addition, their final location may be distant from their point of deposition. Similarly, whereas some pathogens are capable of division within the host, others are incapable of division within the mammalian host (e.g., helminths).
5.4¡ª
Antibody Production in Response to Immunization/Vaccination
Individuals can be rendered resistant to infectious agents by either passive immunization or active immunization. In general, the beneficial effects of immunization are mediated by antibodies, and therefore the effects of immunization can be monitored by the immunoassayist.
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5.4.1¡ª
Passive Immunization.
Passive immunization is accomplished by transferring antibodies from a resistant to a susceptible host. Passively transferred antibodies confer a temporary but immediate resistance to infection, but are gradually catabolized by the susceptible host. Once passive protection wanes, the recipient becomes susceptible to infection again. Passively transferred antibodies can be acquired by the recipient either transplacentally and transcolostrally as in neonates, or by injection of purified antibodies from a resistant donor into a susceptible recipient.
5.4.2¡ª
Active Immunization
Active immunization is accomplished by administering antigens of infectious agents to individuals so that they respond by producing antibodies that will neutralize the infectious disease agents, once contracted. Reexposure to such agents, following active immunization, will result in an anamnestic immune response, in which the antibodies or effector cells produced will be capable of neutralizing the effect of or destroying the inciting agents. Such antibodies are known as protective antibodies, and their complementary antigens as protective antigens. The protection conferred by active immunization is not immediate, as in passive immunization, because the immune system requires considerable time process such antigens and produce protective antibodies. However, the advantage of active immunization is that it is 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 antigenic spectrum of the preparation used for immunization. Protection can be afforded using different approaches in the formulation of vaccines.
5.4.3¡ª
Live Vaccines
Live vaccines may be attenuated by the passage of agents (e.g., viruses) in an unusual host so that they become nonpathogenic to vaccinated animals. Usually these are good vaccines because they supply the same antigenic stimulus as the disease agent. There can be problems of reversion to the pathogenic agent and some replication of the agent usually occurs.
5.4.4¡ª
Modified Vaccines: Whole Disease Agent
Vaccines can be grown and then chemically modified (e.g., heat killed, nucleic acid modified [mutagens], or formaldehyde treated). These are potentially good vaccines in that full antigenic spectrum is given. The antigenic mass must be high since there is no replication to challenge the immune system. Repeat vaccinations are common to elevate antibody levels.
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5.4.5¡ª
Purified Antigens
Protective antigens can be identified and used as proteins, polypeptides, and peptides as immunogens, usually with adjuvants. Usually these vaccines are not as good as those in which total antigenic spectrum is used. They have the advantages of being able to synthesize products on large scale by chemical methods (e.g., as peptides) and are noninfectious.
5.4.6¡ª
DNA Technology Products
Genes producing particular immunogens can be inserted into replicating agents so that their products are expressed. Novel approaches include use of mammalian viruses, insect viruses (baculovirus expression), yeasts, E. coli.
5.4.7¡ª Generalities
Most vaccines are administered by either sc or im injections. When vaccinating large herds of animals, other techniques such as high-pressure jet injections may be employed. Obviously the risk of administering unwanted or contaminating organisms and antigens should be minimal; hence, sterile administration of vaccines is indicated. Subcutaneous or im vaccination should induce all antibody isotypes given the fact that the inciting antigens are capable of doing so. Therefore, the immunoassayist must consider whether total antibody assays, isotype-specific assays, or assays to detect antigen clearance are to be utilized to assess the 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 an aerosolized vaccine (e.g., diseases of the respiratory tract). In these instances, the production of local antibodies to prevent the ingress of pathogens through the GI or respiratory tree barriers should be sought. The immunoassayist must decide whether an assay for isotype-specific antibodies, notably IgA, may provide deeper insight into the benefits of vaccination than an assay for total antibody.
In some instances in which 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.
5.5¡ª
Antibody Production in Response to Infectious Agents
It is beyond the scope of this book to catalog the humoral immune responses produced in mammals in response to the variety of infectious disease agents
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such as viruses, bacteria, fungi, protozoa, helminths, and arthropods. Such information may be obtained from textbooks and specialized review articles. This section deals with the general considerations of hostparasite 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 distant from their point of deposition. Therefore, these pathogens involve multiple organs. 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 be proportional to the number of pathogens at the time of sampling. When pathogens do 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 or low concentrations of antigens and antibodies are to be sought. When antibody titers of less than 1:50 are anticipated, serum dilutions of less than 1:50 or possibly less than 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, complement, basic peptides, and proteins), 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 responses that reduce pathogen numbers by lysis, agglutination or phagocytosis and that reduce the antigen load are normally regarded as protective responses. 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). Because infectious disease agents stimulate antibody production, these antibodies can prove useful to the immunoassayist for detecting exposure to pathogens.
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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. When a variety of antibody isotypes is produced in response to infection, this isotypic variation can be used 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, a direct correlation can be made between ELISA positivity and current infection. When 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) the consensus of opinion indicates that the more specific antigens or 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.
5.5.1¡ª
Effect of Antibody 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 genes carried by viruses varies from 3 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 who is interested 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 of relevant to specific problems.
Because the outer surfaces (capsids) of virus 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 in which viruses are present in plasma and tissue fluids (vector transmitted) or secretory IgA antibodies where viruses are present on epithelial surfaces (air-
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borne, close contact). Some viruses that replicate entirely on epithelial surfaces (e.g., respiratory tree, GI tract, genitourinary [GU] tract) and that do 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.
5.5.2¡ª
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 neutralization of toxins. 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 antibodies are regarded as more effective opsonins than IgM in the absence of complement than IgG. It is the most effective antibody isotype in the presence of complement. Thus, IgM antibodies are more effective in inducing complement-mediated lysis and bacterial opsonization prior to phagocytosis. Antibody can block transport mechanisms and bacterial receptors (e.g., for iron-chelating compounds); neutralize immunorepellants (which interfere with normal phagocytosis), and neutralize spreading factors that facilitate invasion (e.g., enzymes, hyaluronidase).
5.5.3¡ª
Effect of Antibody in Protozoan Infection