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55 Cards in this Set
- Front
- Back
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Q: Where in the heavy and light chains do we find the variable regions?
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-Both light and heavy chains have variable regions which are found within the most amino terminal domain of each chain.
-We know that the variable region binds antigen from two lines of evidence: one indicating that Fab pieces of IgG were would bind antigen, and the other that the Fab pieces had variation in their amino acid sequences. . |
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Q: How was it determined that there was an antigen-binding component on the Ig?
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-Proteolytic digestion of immunoglobulin indicated an antigen binding component and that it was heterogeneous (because it would not crystallize). (Only pure proteins, without amino acid variations, will crystallize from solution. Mixtures of proteins will not crystallize.)
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Q: Describe the antigen-binding fragment (Fab).
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-has varying amino acid sequences and fails to crystallize. The Fab contains an intact L chain disulfide bonded to a piece of H chain. One Fab binds only one epitope. The Fc (crystallizable fragment) contains two identical proteolytic ally cut pieces of H chain.
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Q: What happens to the heavy chain when given pepsin?
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-Digestion with the protease pepsin cleaves the carboxy terminal regions of heavy chains into small pieces, and cuts only on the carboxy terminal side of the disulfide bonds that interconnect the H chains. Thus pepsin digestion generates an F(ab')2 fragment that is still disulfide bonded between the H chains. The F(ab')2 can bind two antigenic epitopes. Fab fragments can not create lattices, but F(ab')2 fragments can. Destroys Fc (cyrstallizable fragment)
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Q: What happens when it is digested with papain?
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-gives Fab and Fc fragments, papain digestion allows separation of two antigen-binding regions (the Fab fragments) from the portion of the IgG molecule that activates complement and binds to Fc receptors
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Q: What information did digestion give concerning the amino acid sequences?
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-Hyper variable regions of immunoglobulins were detected within the L and H chain variable domains. It was immediately recognized that this variation would contribute to different "locks' to bind antigen. the hypervariable regions were also termed complementarity determining regions (CDRs)
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Q: What happens to Ig secretion from B cells as B cell responses to particular antigens mature?
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-We now know that as B cell responses to particular antigens mature, the B cells switch from production of IgM to production of IgG, IgA or IgE. When this isotype switch happens within a single cell or a clone of progeny originating from one cell, the L chains and the variable regions of the H chains are retained, but the H chain constant domains are switched from one Ig to another class and are therefore different.
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Q: Describe the coding for the kappa and lambda chains.
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-The 2 kinds of light chains, kappa and lambda, are encoded at 2 different loci in the genome.
-Kappa light chains represent about half of all human immunoglobulin (Ig) and lambda light chains the other half of all human Ig. -Both light chains are found in all classes and subclasses of Ig. |
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Q: Describe the classification of the different Ig classes.
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-Immunoglobulin classes have different heavy chains and use the same (kappa or lambda) light chains. The classes are named with capital Roman letters and the heavy chains with the corresponding Greek lower case letters, i.e., IgA, IgD, IgE, IgG, & IgM have alpha, delta, epsilon, gamma and mu heavy chains.
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Q: Which Igs have subclasses?
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-IgA and IgG
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Q: What happens when anumals are immunized with Ig of a different species?
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-When animals are immunized with Ig of a different species, the resulting antisera will have anti-light chain antibodies and additional antibodies that react only with one of the different heavy chain classes or their subclasses. On the basis of this observation (that all proteins in a class or subclass have heavy chain antigens in common), the classes and subclasses of Ig are also termed Ig isotypes. In addition, B lymphocytes use membrane forms of the different Ig classes as the B cell surface receptors for antigen.
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Q: What immunochemical features does IgM have?
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-Mu heavy chains with one variable domain and 4 (not 3) constant domains. Human IgM has no subclasses.
- Pentameric repeat of the monomer unit and therefore 10 antigen binding sites. The pentameric units are held together by disulfide inter-unit bonds. -One 15,000 Dalton J chain per molecule of IgM that is considered necessary for formation of immunoglobulin multimeric Ig molecules. The J chain has been likened to a "clasp" to hold the 5 Ig molecules together. -M.wt. = ~900,000 |
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Q: Describe the biological features of IgM.
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-a concentration of ~ 1.2 mg/ml in serum and a half life in serum of ~5 days. IgM fixes complement well and is very important in clearing bacteria and yeast from the blood. One molecule of IgM bound to antigen will fix complement and kill a pathogenic bacterium
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Q: What are the distinct immune response properties of IgM?
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-IgM has distinct immune response properties. IgM is the first antibody made after antigen administration, appearing in serum about 5 days after antigen administration. See yesterday's figure 1 for the time course of IgM and IgG. Usually, these IgM antibodies are replaced by IgG antibodies to the same antigen at about 1 0 days after antigen administration.
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Q: What routes of immunization elicit IgM release?
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-IgM is elicited by antigens introduced by several routes of immunization: Some antigens, such as the repeated carbohydrate antigens of yeast and bacteria, elicit primarily IgM responses and very little IgG. These repeated antigens are also the so-called T cell-independent antigens
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Q: What are the four distinct subclasses of IgG?
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-IgG1 - IgG2
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Q: Describe the immunochemical features of IgG.
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-Gamma heavy chains with one variable and 3 constant domains.
-A monomeric unit that has 2 antigen binding sites per molecule. -Four human IgG subclasses termed IgGl, IgG2, IgG3 and IgG4 which differ markedly in the disulfide bridges between the two heavy chains. -M.wt. = ~150,000 |
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Q: Describe the biological features of IgG.
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-10 mg/mL in serum (serum is about 70 mg/ml total protein)
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Q: How long does IgG response last?
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-Immune response properties of IgG confer long lasting antibody reactivity to serum.
-IgG antibody responses follow IgM antibody responses when antigens are administered systemically or intramuscularly. IgG and IgM are both very important in clearing foreign antigens from the body. Serum IgG antibody responses can persist for years. IgG3 and IgG1 both fix complement well. |
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Q: What makes IgG unique in terms of passively acquired immunity?
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-IgG is the only immunoglobulin class to cross the placenta and is therefore responsible for passively acquired immunity of the newborn, which will decrease with time due to the IgG half life. The infant will not make its own antibodies to these antigens until it encounters them as immunogens
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Q: What routes of immunization elicit IgG?
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-Antigens recognized by IgG and routes of immunization that elicit IgG: IgG is produced to almost all antigens that enter intramuscularly or systemically. IgG production continues long after the antigen has disappeared. This continuous Ig production is what makes vaccination feasible.
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Q: What role does IgG have on effector cells?
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-Effector cells cac mediate immune functions by using membrane receptors for IgG when IgG antibodies are bound to bacteria, yeast or cells.
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Q: What do macrophages and neutrophils use their IgG receptors for?
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-Macrophages and neutrophils use their IgG receptors in processes to ingest and kill bacteria and yeast which have bound specific antibodies.
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Q: What role does IgG have with NK cells?
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-Natural killer (NK) cells are lymphocytes which can kill cells infected with viruses. They can also directly kill certain types of leukemic tumor cells. Furthermore, NK cells will mediate IgG antibody-dependent cell-mediated cytotoxicity (ADCC) of any cell coated with specific IgG antibody. NK cells are different from T and B cells.
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Q: What are the requirements for ADCC (antibody-dependent cell-mediated cytotoxicity)?
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-The requirements are target cells with antigens, antibody of the cognate antigen specificity and appropriate Ig isotype, and killer cells with Fe receptors for the Ig lsotype.
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Q: What are the requirements for NK cell mediated ADCC?
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-Target cells with membrane-associated antigens. (Measles virus antigen in the example below.) The target cells are labeled with radioactive 51Cr in their cytoplasm, which is released when the cells die and their plasma membranes are disrupted.
-Antibody to the target cell antigens. (IgGl or IgG3 Ab to measles proteins.) -NK lymphocytes. (From the peripheral blood of any healthy donor. The lymphocyte donor does not have to be immune to measles.) |
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Q: How is the release of intracellular components by the action of killer cells determined?
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-via 51Cr release assay. target cells are labeled by incubation with Na2 51CrO4, killer cells are then added to the labeled target cells, after incubation, the amount of intracellular and extracellular label remaining is determined by counting the radioactivity in the supernate and/or centrifuged cells, the percentage of specific lysis is calculated
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Q: Describe the experimental design with controls.
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-Positive controls produce the desired result (killing).
-Negative controls give values for nonspecific results (no antibody |
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Q: How is % specific release calculated?
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-as [(Expt'l - Spontaneous release)/(Detergent max - Spontaneous release)] x 100
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Q: Describe the immunochemical features of IgA.
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-An alpha heavy chain with one variable and 3 constant domains.
-Monomers or multimers of 2-3 repeating units. Each multimer contains one J chain, with proportional numbers of antigen binding sites. -The human has two subclasses IgAl and IgA2. -Much IgA is secreted and found at mucus membranes. Secretory IgA is a dimer with one J chain (the same J of the clasp of IgM) and one secretory component protein. The secretory component (SC) is not made by lymphocytes, but made by epithelial cells at the mucosa. Serum IgA has the J piece if it is multimeric but does not have the secretory component protein. -Secretory IgA with the J chain and secretory component has a M.wt. of ~400,000. |
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Q: What are the biological features of IgA?
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-IgA is found in serum and in secretions, including lacrimal, nasal, salivary, intestinal, mammary, and genitourinary secretions. Serum IgA has a concentration of ~2 mg/ml and a half life of ~6 days. IgA prevents pathogens from adhering to the mucosal membranes and reduces their infectivity.
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Q: What are the immune response properties of IgA?
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-Because of circulation and homing of IgA-producing B cells to mucosal areas, secretions will have IgA antibodies to pathogens that entered at another site. Thus IgA-producing B cells from the gut can home to the mammary gland so that the IgA antibodies in human milk provide passive protection to the newborn against gut-associated pathogens. In the gut, IgA antibodies bind to bacteria and viruses and prevent them from adhering to the gut wall or crossing into the body. The bacteria and viruses are rapidly "flushed" out of the body. In the human, none of the immunoglobulins cross the infant's gut barrier. Maternal IgA stays in the baby's gut and protects against enteric pathogens. Breast feeding of infants should be strongly encouraged, in this country as well as in underdeveloped countries. IgA does not fix complement.
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Q: What routes of immunization elicit IgA?
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-Most antigens that enter via mucus membranes or cross the gut elicit IgA antibody responses. These include antigens of the normal bacterial flora, pathogenic bacteria and viruses and Giardia. You will lean where the IgA-producing lymphoid tissues are in the lymphoid organs lecture.
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Q: What does IgE look like?
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-just like IgG, is a monomeric unit
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Q: What are the immunochemical feature of IgE?
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-IgE has epsilon heavy chain with one variable and 4 (not 3) constant domains.
-IgE a monomer Ig unit that has 2 antigen binding sites per molecule. -There are no IgE subclasses in the humans -M.Wt = ~190,000 |
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Q: What are the biological features of IgE?
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-IgE is found in serum, but also and very importantly fixed on mast cells and basophils in the body. Serum concentrations are VERY low, about 0.5 micrograms per mL. These concentrations are too low to detect by immunodiffusion or other standard means. Radioimmunoassays are usually used to detect IgE. The half life of IgE in serum is fast, estimated as fast as 1 day and ranging between 1-5 days.
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Q: What Ig mediates immediate hypersensitivey?
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-Only IgE mediates immediate hypersensitivity because only IgE binds to mast cells and basophils, "Atopy" means allergy. An "atopic" person is prone to allergies. Urticaria or hives, allergic asthma, and death due to suffocation by unarrested anaphylaxis are all manifestations of immediate hypersensitivity.
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Q: Describe the relationship between mast cells or basophils and IgE.
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-Each mast cell or basophil has IgE bound to high affinity membrane F c epsilon receptors that bind only IgE. There are about 40,000 IgE Fc receptors per mast cell. Each mast cell can hold IgE antibodies specific for many different antigens. Therefore, each mast cell has the potential to respond to hundreds of different antigens by exocytosing granules. The preloading of Fc2e mast cells receptors with IgE greatly enhances the probability of any antigen that elicited IgE stimulating any mast cell or basophil.
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Q: Describe why mast cells are considered sentinel cells.
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-Mast cells are considered "sentinel" cells of infection and inflammation that announce the presence of pathogens. They are immobile cells found throughout the interfaces with the environment. There are two predominant types of mast cells: "mucosal" mast cells found in the oral and gastric mucosa and "connective tissue" mast cells that are found in the skin, around the capillary venules, in the lungs, and in connective tissue. Mast cells can respond to either of two stimuli: to antigens that bind IgE or to the complement anaphylatoxins. Basophils, a subset of polymorphonuclear leukocytes, circulate in the blood, also bind IgE, and can release similar mediators upon antigen stimulation.
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Q: What are the immune response properties of IgE?
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-the Ig involved in allergy or the “reagenic” Ab
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Q: What are the sequence of reactions following entry of antigen for IgE?
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-Antigen binds to IgE antibody on the mast cells.
-The antigen must crosslink two or more Fce receptors by binding to two or more IgE molecules. Therefore, the antigen must have at least two epitopes. To crosslink the receptors of one mast cell, these epitopes can be the same or different since the mast cell has numerous antibodies. Remember, most antigens have several different epitopes and sometimes several copies of the same epitope. Consider the number of different and repeated ewes that are found on a grain of pollen. -When two or more IgE molecules on the mast cell have bound the same antigen and Fce receptors are brought into close proximity, a signal is transduced to the interior of the mast cell. The mast cell then releases (exocytoses) some of its granules. |
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Q: What are the factors released by the mast cells?
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-The mast cells release many mediators of immediate hypersensitivity that result in edema, itching, pain, and local coagulation. There are three means to produce mediators:
(1) immediate release of granule contents (2) rapid arachidonic acid metabolism to produce lipid mediators |
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Q: Why did immediate hypersensitivity evolve?
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-It is thought that immediate hypersensitivity evolved to kill parasitic worms, like Ascaris. The overall effect is to block drainage from the site and recruit eosinophils which are the cells capable of killing the worms. Eosinophils can kill by IgE-mediated antibody-dependent cell-mediated cytotoxicity (ADCC).
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Q: When is IgE elevated?
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-High serum IgE correlates with certain parasitic diseases and ' aspergillosis. However, in common practice, a clinician is interested in" measuring IgE antibodies specific for particular allergens.
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Q: How is IgE and IgE Ab detected?
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-Total IgE in serum is measured by enzyme-linked immurtosorbant assays (ELISAs) which will be described Friday. There is about 1/4 microgram/mL IgE in the serum of normal individuals, which is tow low to detect by radial immunodiffusion.
-IgE antibodies to particular antigens can be assayed by skin tests or by serum tests. |
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Q: Describe skin tests to measure IgE.
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-Skin tests introduce antigens directly to the patient. First a needle prick introduces minute amounts of antigen. . Then if the first test is negative, the clinician follows up with intracutaneous tests. SKIN TESTS CAN INTRODUCE RISKS OF SYSTEMIC ANAPHYLAXIS! The advantages are that the test screens the IgE bound to the patient's mast cells. Serum IgE is not always a reflection of mast cell-bound IgB. IgB can be bound immediately after secretion by nearby plasma cells. On the other hand, skin tests will not necessarily reflect IgE bound to lung mast cells.
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Q: Describe the use of RAST (radioallergosorbent test) in measuring IgE Abs.
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-The antigen is fixed irreversibly onto beads or paper. The papers are mixed with the patient's test serum and then washed to remove any antibodies which didn't bind to the antigen on the paper. Then radioactive 125I rabbit anti-human IgE is added to reaction. The papers are washed to remove any 125I antibody not bound to IgE and then counted for radioactivity. At the end of the assay, counts on the paper indicate that the patient has IgE antibody specific for the antigen on paper. The advantages of this test are that many tests can be run from one blood sample and the patient suffers no discomfort. The disadvantages are that the crucial IgE antibody may be only on the patient's mast cells and not in his serum.
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Q: Describe the shape of IgD.
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-IgD is a monomeric unit similar to IgG
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Q: What are the immunochemical features of IgD?
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-IgD has delta heavy chain with one variable domain and 3 constant domains
-IgD is a monomeric unit with 2 antigen binding sites. -In the human there are no IgD subclasses. -M.wt. =~180,000 |
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Q: What are the biological features of IgD?
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-A membrane form of IgD is found with IgM in the plasma membranes of "virgin" B cells that have never encountered their antIgens. IgD has very low serum concentrations of ~30 micrograms/rot and a serum half life of ~3 days.
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Q: Describe the immune response properties of IgD.
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-IgD has no known function. Its presence in virgin B cell membranes has led to suggestions that it may modulate B cell responses. However, IgD gene 'knock-out' mice have antibody responses similar to wild type (IgD normal) mice.
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Q: What are the routes of immunizations that activate IgD?
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-Unknown
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Q: Define affinity.
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Affinity is a measurement of the binding strength of a single Fab antigen-binding site for its single antigenic epitope, expressed as the reciprocal of the concentration of a hapten that will bind half the antibody binding sites.
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Q: Define avidity.
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-Avidity is a measurement of the tota1 combined binding strength of a complete immunoglobulin molecule for a complex antigen that contains repeating epitopes. Thus an IgM antibody molecule with low affinity for a particular antigen (but that has 10 antigen binding sites) will bind with high avidity to a complex antigen that contains repeats of the epitope.
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Q: What are some medical applications for immunoglobulins?
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-The classes of immunoglobulins have different functions in the great war against pathogens
-IgE has a role in allergy. In the management of allergies, you may want to produce IgG antibodies to allergenic antigens. These IgG antibodies do not bind to mast cells, do not trigger allergic responses and will compete with IgE for the antigens. -You may use IgG antibody preparations, like anti-hepatitis A IgG to passively immunize travelers who will be at risk of exposure to hepatitis A in geographic regions of poor sanitation. |
