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In the initial stages of the immune response, small numbers of immature B lymphocytes are able to bind foreign antigen molecules weakly via the antibodies expressed on their
surfaces. Each plasma cell secretes a single antibody with high affinity for antigen. Plasma cells are virtual antibody factories that can be identified in electron micrographs by their extensive rough endoplasmic reticulum. The scope of antibody diversity
is immense - vertebrates are capable of producing billions of antibody molecules with distinct specificities.
Polyclonal vs. monoclonal antibodies
For our western blots, we will be using both monoclonal and polyclonal antibodies. As their names imply, monoclonal antibodies bind to the same epitope on an antigen. Polyclonal antibodies are actually mixtures of antibodies that bind to different epitopes on an antigen. An animal’s response to antigen is polyclonal, because antigens stimulate the proliferation of multiple lymphocyte clones, each of which produces a different antibody to the antigen. Consequently, the serum collected from an immunized animal contains a mixture of antibodies with different specificities. The polyclonal antibodies used in the lab are purified from the sera of animals that have been inoculated with antigen.
By contrast, monoclonal antibodies are produced in the lab from cultured hybridoma cells. Hybridoma cells are generated by fusing a lymphocyte from an immunized animal, most commonly a mouse, with a cancerous myeloma cell that can divide indefinitely in culture (right). Because the lymphocytes from the spleen of an immunized mouse recognize a range of different epitopes on an antigen, the hybridomas resulting from the fusion secrete a variety of different antibodies. Standard culture techniques are then used to isolate individual hybridoma cell lines, each of which secretes a unique antibody that binds to a single epitope.
Hybridoma technology has revolutionized biomedical research since its description (Kohler & Milstein, 1975), both because monoclonal antibodies recognize well-defined epitopes and because monoclonal antibodies can be produced indefinitely by cultured hybridoma cells. Investigators often use both monoclonal and polyclonal antibodies at different steps in western blots.
In this section, you will explore the following questions:
- What is cross-reactivity?
- What is the basic structure of an antibody, and what are the functions of antibodies?
- How are antibodies produced?
Connection for AP ® Courses
Much of the information in this section is not within the scope for AP ® . Antibodies, also known as immunoglobulins, are proteins produced and secreted by plasma cells (differentiated B lymphocytes) that mediate the humoral immune response. Antibodies are Y-shaped proteins consisting of four polypeptides with at least two binding sites for a specific antigen. The areas where the antigen is recognized on the antibody are variable domains. For AP ® , you do not need to know the different classes of antibodies or the molecular structure of a specific antibody. What is important to understand is that antibodies are antigen-specific. When antibodies bind antigens, they can neutralize pathogens, mark them for phagocytosis, or activate the complement cascade. Because secreted antibodies can remain in the circulation for many years, secondary exposure to a pathogen results in a faster immune response. Antibodies occur in the blood, in gastric and mucus secretions, and in breast milk, thus providing passive immunity to the infant.
Information presented and the examples highlighted in the section support concepts outlined in Big Idea 2 and Big Idea 4 of the AP ® Biology Curriculum Framework. The AP ® Learning Objectives listed in the Curriculum Framework provide a transparent foundation for the AP ® Biology course, an inquiry-based laboratory experience, instructional activities, and AP ® exam questions. A learning objective merges required content with one or more of the seven science practices.
|Big Idea 2||Biological systems utilize free energy and molecular building blocks to grow, to reproduce, and to maintain dynamic homeostasis.|
|Enduring Understanding 2.D||Growth and dynamic homeostasis of a biological system are influenced by changes in the system’s environment.|
|Essential Knowledge||2.D.4 Plants and animals have a variety of chemical defenses against infections that affect dynamic homeostasis.|
|Science Practice||1.1 The student can create representations and models of natural or man-made phenomena and systems in the domain.|
|Science Practice||1.2 The student can describe representations and models of natural or man-made phenomena and systems in the domain.|
|Learning Objective||2.30 The student can create representations or models to describe nonspecific immune defenses in animals.|
|Big Idea 4||Biological systems interact, and these systems and their interactions possess complex properties.|
|Enduring Understanding 4.C||Naturally occurring diversity among and between components within biological systems affects interactions with the environment.|
|Essential Knowledge||4.C.1 Variation in molecular units provides cells with a wider range of functions.|
|Science Practice||6.2 The student can construct explanations of phenomena based on evidence produced through scientific practices.|
|Learning Objective||4.22 The student is able to construct explanations based on evidence of how variation in molecular units provides cells with a wider range of functions.|
An antibody molecule is comprised of four polypeptides: two identical heavy chains (large peptide units) that are partially bound to each other in a “Y” formation, which are flanked by two identical light chains (small peptide units), as illustrated in Figure 33.22. Bonds between the cysteine amino acids in the antibody molecule attach the polypeptides to each other. The areas where the antigen is recognized on the antibody are variable domains and the antibody base is composed of constant domains.
In germ-line B cells, the variable region of the light chain gene has 40 variable (V) and five joining (J) segments. An enzyme called DNA recombinase randomly excises most of these segments out of the gene, and splices one V segment to one J segment. During RNA processing, all but one V and J segment are spliced out. Recombination and splicing may result in over 10 6 possible VJ combinations. As a result, each differentiated B cell in the human body typically has a unique variable chain. The constant domain, which does not bind antibody, is the same for all antibodies.
Similar to TCRs and BCRs, antibody diversity is produced by the mutation and recombination of approximately 300 different gene segments encoding the light and heavy chain variable domains in precursor cells that are destined to become B cells. The variable domains from the heavy and light chains interact to form the binding site through which an antibody can bind a specific epitope on an antigen. The numbers of repeated constant domains in Ig classes are the same for all antibodies corresponding to a specific class. Antibodies are structurally similar to the extracellular component of the BCRs, and B cell maturation to plasma cells can be visualized in simple terms as the cell acquires the ability to secrete the extracellular portion of its BCR in large quantities.
Antibodies can be divided into five classes—IgM, IgG, IgA, IgD, IgE—based on their physiochemical, structural, and immunological properties. IgGs, which make up about 80 percent of all antibodies, have heavy chains that consist of one variable domain and three identical constant domains. IgA and IgD also have three constant domains per heavy chain, whereas IgM and IgE each have four constant domains per heavy chain. The variable domain determines binding specificity and the constant domain of the heavy chain determines the immunological mechanism of action of the corresponding antibody class. It is possible for two antibodies to have the same binding specificities but be in different classes and, therefore, to be involved in different functions.
After an adaptive defense is produced against a pathogen, typically plasma cells first secrete IgM into the blood. BCRs on naïve B cells are of the IgM class and occasionally IgD class. IgM molecules make up approximately ten percent of all antibodies. Prior to antibody secretion, plasma cells assemble IgM molecules into pentamers (five individual antibodies) linked by a joining (J) chain, as shown in Figure 33.23. The pentamer arrangement means that these macromolecules can bind ten identical antigens. However, IgM molecules released early in the adaptive immune response do not bind to antigens as stably as IgGs, which are one of the possible types of antibodies secreted in large quantities upon re-exposure to the same pathogen. Figure 33.23 summarizes the properties of immunoglobulins and illustrates their basic structures.
IgAs populate the saliva, tears, breast milk, and mucus secretions of the gastrointestinal, respiratory, and genitourinary tracts. Collectively, these bodily fluids coat and protect the extensive mucosa (4000 square feet in humans). The total number of IgA molecules in these bodily secretions is greater than the number of IgG molecules in the blood serum. A small amount of IgA is also secreted into the serum in monomeric form. Conversely, some IgM is secreted into bodily fluids of the mucosa. Similar to IgM, IgA molecules are secreted as polymeric structures linked with a J chain. However, IgAs are secreted mostly as dimeric molecules, not pentamers.
IgE is present in the serum in small quantities and is best characterized in its role as an allergy mediator. IgD is also present in small quantities. Similar to IgM, BCRs of the IgD class are found on the surface of naïve B cells. This class supports antigen recognition and maturation of B cells to plasma cells.
Differentiated plasma cells are crucial players in the humoral response, and the antibodies they secrete are particularly significant against extracellular pathogens and toxins. Antibodies circulate freely and act independently of plasma cells. Antibodies can be transferred from one individual to another to temporarily protect against infectious disease. For instance, a person who has recently produced a successful immune response against a particular disease agent can donate blood to a nonimmune recipient and confer temporary immunity through antibodies in the donor’s blood serum. This phenomenon is called passive immunity it also occurs naturally during breastfeeding, which makes breastfed infants highly resistant to infections during the first few months of life.
Antibodies coat extracellular pathogens and neutralize them, as illustrated in Figure 33.24, by blocking key sites on the pathogen that enhance their infectivity (such as receptors that “dock” pathogens on host cells). Antibody neutralization can prevent pathogens from entering and infecting host cells, as opposed to the CTL-mediated approach of killing cells that are already infected to prevent progression of an established infection. The neutralized antibody-coated pathogens can then be filtered by the spleen and eliminated in urine or feces.
Antibodies also mark pathogens for destruction by phagocytic cells, such as macrophages or neutrophils, because phagocytic cells are highly attracted to macromolecules complexed with antibodies. Phagocytic enhancement by antibodies is called opsonization. In a process called complement fixation, IgM and IgG in serum bind to antigens and provide docking sites onto which sequential complement proteins can bind. The combination of antibodies and complement enhances opsonization even further and promotes rapid clearing of pathogens.
Affinity, Avidity, and Cross Reactivity
Not all antibodies bind with the same strength, specificity, and stability. In fact, antibodies exhibit different affinities (attraction) depending on the molecular complementarity between antigen and antibody molecules, as illustrated in Figure 33.25. An antibody with a higher affinity for a particular antigen would bind more strongly and stably, and thus would be expected to present a more challenging defense against the pathogen corresponding to the specific antigen.
The term avidity describes binding by antibody classes that are secreted as joined, multivalent structures (such as IgM and IgA). Although avidity measures the strength of binding, just as affinity does, the avidity is not simply the sum of the affinities of the antibodies in a multimeric structure. The avidity depends on the number of identical binding sites on the antigen being detected, as well as other physical and chemical factors. Typically, multimeric antibodies, such as pentameric IgM, are classified as having lower affinity than monomeric antibodies, but high avidity. Essentially, the fact that multimeric antibodies can bind many antigens simultaneously balances their slightly lower binding strength for each antibody/antigen interaction.
Antibodies secreted after binding to one epitope on an antigen may exhibit cross reactivity for the same or similar epitopes on different antigens. Because an epitope corresponds to such a small region (the surface area of about four to six amino acids), it is possible for different macromolecules to exhibit the same molecular identities and orientations over short regions. Cross reactivity describes when an antibody binds not to the antigen that elicited its synthesis and secretion, but to a different antigen.
Cross reactivity can be beneficial if an individual develops immunity to several related pathogens despite having only been exposed to or vaccinated against one of them. For instance, antibody cross reactivity may occur against the similar surface structures of various Gram-negative bacteria. Conversely, antibodies raised against pathogenic molecular components that resemble self molecules may incorrectly mark host cells for destruction and cause autoimmune damage. Patients who develop systemic lupus erythematosus (SLE) commonly exhibit antibodies that react with their own DNA. These antibodies may have been initially raised against the nucleic acid of microorganisms but later cross-reacted with self-antigens. This phenomenon is also called molecular mimicry.
Antibodies of the Mucosal Immune System
Antibodies synthesized by the mucosal immune system include IgA and IgM. Activated B cells differentiate into mucosal plasma cells that synthesize and secrete dimeric IgA, and to a lesser extent, pentameric IgM. Secreted IgA is abundant in tears, saliva, breast milk, and in secretions of the gastrointestinal and respiratory tracts. Antibody secretion results in a local humoral response at epithelial surfaces and prevents infection of the mucosa by binding and neutralizing pathogens.
The structure of an antibody is similar to the extracellular component of which receptor?
The first antibody class to appear in the serum in response to a newly encountered pathogen is ________.
What is the most abundant antibody class detected in the serum upon reexposure to a pathogen or in reaction to a vaccine?
Breastfed infants typically are resistant to disease because of ________.
What are the benefits and costs of antibody cross reactivity?
Cross reactivity of antibodies can be beneficial when it allows an individual's immune system to respond to an array of similar pathogens after being exposed to just one of them. A potential cost of cross reactivity is an antibody response to parts of the body (self) in addition to the appropriate antigen.
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Producing Polyclonal Antibodies
Antibodies used for research and diagnostic purposes are often obtained by injecting a lab animal such as a rabbit or a goat with a specific antigen. Within a few weeks, the animal’s immune system will produce high levels of antibodies specific for the antigen. These antibodies can be harvested in an antiserum, which is whole serum collected from an animal following exposure to an antigen. Because most antigens are complex structures with multiple epitopes, they result in the production of multiple antibodies in the lab animal. This so-called polyclonal antibody response is also typical of the response to infection by the human immune system. Antiserum drawn from an animal will thus contain antibodies from multiple clones of B cells, with each B cell responding to a specific epitope on the antigen (Figure 2).
Figure 2. This diagram illustrates the process for harvesting polyclonal antibodies produced in response to an antigen.
Lab animals are usually injected at least twice with antigen when being used to produce antiserum. The second injection will activate memory cells that make class IgG antibodies against the antigen. The memory cells also undergo affinity maturation, resulting in a pool of antibodies with higher average affinity. Affinity maturation occurs because of mutations in the immunoglobulin gene variable regions, resulting in B cells with slightly altered antigen-binding sites. On re-exposure to the antigen, those B cells capable of producing antibody with higher affinity antigen-binding sites will be stimulated to proliferate and produce more antibody than their lower-affinity peers. An adjuvant, which is a chemical that provokes a generalized activation of the immune system that stimulates greater antibody production, is often mixed with the antigen prior to injection.
Antiserum obtained from animals will not only contain antibodies against the antigen artificially introduced in the laboratory, but it will also contain antibodies to any other antigens to which the animal has been exposed during its lifetime. For this reason, antisera must first be “purified” to remove other antibodies before using the antibodies for research or diagnostic assays.
Until the development of an effective vaccine, the results from the clinical trials evaluating the therapeutic alternatives described above are urgently needed. This will be essential for the care of patients, who could increase in number during in the possible following outbreaks, before we have a resolving vaccine.
The immune system taught us how to defend ourselves from the viruses, and the vaccines taught the most extraordinary lesson, since the days of Jerne’s smallpox vaccine (1796). The 1984 Nobel Prize for Medicine Niels Kaj Jerne “poetically” described the immune system as the mirror image of the universe, and the vaccines exploit this amazing capability, in order to elicit the protective immune responses against every small fragment of any pathogen outside of us (“non-self” antigens).
Watch the video: Ένζυμα - βιολογικοί καταλύτες (November 2022).