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Biol 430 Question Bank Antibodies 1. In this diagram of an antibody molecule identify the components labeled as: A. _________ B. _________ C. _________ D. _________ E. _________ F. _________ G. _________ H. _________ I. _________ J. _________ 2. This Figure shows the genetic recombination of Ig Heavy Chain genes and subsequent processing of the transcript in a mature B-cell. 1. Identify the genetic elements identified as A1: A2: A3: A4: 2. Briefly describe what is happening in the steps labeled as ‘B’. 3. What is happening in step ‘C’? 4. What are the two products shown as ‘D’ Biol 430 Question Bank Antibodies Page 1 3. Some questions about regulation of gene segment rearrangements… A. Through the regulatory process called __________________, rearrangement and expression of antibody genes is restricted to a single locus at a time. Why is this essential for the proper functioning of mature B-cells? B. How many chances does a B-cell have to make a functional heavy chain gene? Explain. C. How many chances to make a functional light chain gene? Explain. 4. Using the following estimates of the number of antibody gene segments in mice, and the following questions: H chain V D J κ light chain γ light chain Estimated number of segments 200 300 3 15 0 0 4 4 3 A. What is the number of possible VDJ combinations for the heavy, kappa and lambda chains? B. What is the total number of possible of heavy and light chain combinations? Explain. C. What are three other mechanisms that will contribute to the total diversity of antibodies produced? D. Why will only a small percentage of the B-cells be released from the bone marrow? What will happen to the rest? Biol 430 Question Bank Antibodies Page 2 5. The figure to the right shows the steps of a classic experiment performed by Tonegawa and Hozumi in 1976. In this experiment DNA from normal mouse embryo cells and from a plasmacytoma were used. A plasmacytoma (another name for a myeloma) is a malignancy in which there is a proliferation of a single plasma cell clone which produces a single type of antibody. DNA from the two cell types were cleaved with a DNA restriction enzyme, the DNA fragments were separated by size using electrophoresis and transferred to nitrocellulose. The nitrocellulose was then probed with radiolabeled RNA complementary to the heavy chain rearranged constant region of the plasmacytoma. The lower diagram shows how the probe would bind to a DNA fragment containing the embryonic constant region gene. The autoradiogram shows that the probe bound to a single DNA fragment from embryonic cells, but to two fragments from the plasmacytoma. A. Why does a RNA probe bind to only certain DNA fragments? B. Why is the DNA fragments transferred to nitrocellulose after separation by gel electrophoresis? C. Explain why the probe binds to one DNA fragment from embryonic cells but two DNA fragments from the plasmacytoma cells. D. Was HC allele rearrangement successful? Explain. Biol 430 Question Bank Antibodies Page 3 6. In 1994, Lonberg et al. (Nature 368:856) reported the creation of transgenic mice that produced human antibodies. They started with a strain of mouse in which the heavy chain and light chain loci had been ‘knocked-out’ (made non-functional). They then introduced into embryos from these mice genes for the heavy and light chains from humans; transgenic mice were then grown from these embryos. When exposed to antigens, these mice produce human antibodies against the antigens. A. Why was it necessary to start with Ig knock-out mice? B. The antibodies produced from the transgenic mice are ‘humanized’ antibodies; how do these differ from the antibodies from normal mice? C. Suppose the transgenic mice were inoculated with a human protein; would they respond to that protein differently than normal mice? Explain. 7. Match each of the following terms with its best description. Term Description: a. ___ IgA 1. DNA sequences that flank variable region gene segments b. ___ RAG 2. new bases added to a gene segment junction c. ___ IgM 3. most abundant form of antibody found in the blood d. ___ JDV 4. antibody isotype of uncertain function e. ___ IgE 5. enzyme required for antibody gene segment recombination f. ___ mu (μ) 6. blood protein fraction containing antibodies g. ___ IgG 7. arrangement of heavy and light chains in secreted IgM h. ___ JV 8. one of the light chain constant regions i. ___ RSS 9. form of antibody common in breast milk and digestive tract j. ___ IgD 10. example of a Ig heavy chain k. ___ P-nucleotides 11. gene segments found in the heavy chain variable region l. ___ Lambda (λ) 12. antibody class associated with hypersensitivities m. ___ γ-globulin 13. gene segments found in the light chain variable region n. ___ Igα/Igβ 14. additional proteins found in B-cell receptor o. ___ H10L10 15. Ig class of the B-cell receptor Biol 430 Question Bank Antibodies Page 4 8. It is possible to produce antisera (antibodies) against other antibodies or parts of antibodies. Generally this is done by injecting the antibodies (or molecular parts thereof) from one animal species into a different species, which then reacts against the antigen as a foreign substance. Suppose antisera were produced against various molecular components (listed at the top of this table); indicate whether each type of antisera would contain antibodies (+) or not (--) against the molecules listed in the left hand column. Antisera produced against_____________ γ κ IgG IgG μ Chain Chain Fab Fc Chain gamma chain kappa chain whole IgG IgM Fc fragment IgM Fab Fragment 9. As an enthusiastic young investigator you have isolated a protein that you believe is a new type of human immunoglobin (“IgF”), and you undertake a series of experiments to test this hypothesis. A. You find that the protein possesses Ig domains. Is this enough to confirm that this is a type of immunoglobin? Explain. B. You test the ability of the protein to bind to various types of bacteria, but see no reaction. Is this enough to confirm that this is not an immunoglobin? Explain. C. If you were to prepare antisera against human Gamma heavy chain and Kappa light chain, which antisera would contain antibodies that would react with “IgF” if it is indeed an immunoglobin? Explain. 10. Complete the table below pertaining to the properties of IgG and its various subcomponents. Indicate if the molecule (or part) has the property (+) or not (– ). Property Can bind antigen Bivalent antigen binding Binds to Fc receptors Activates complement in presence of antigen Has V domains Has C domains Biol 430 Question Bank whole H IgG chain λ chain Fab Antibodies F(ab)2 Fc Page 5 11. IgG antibodies occur in several subclasses, although the particular effector functions of each subclass are not well understood. The clinical response to antibody-antigen complex is mediated by receptors that bind to the Fc region of the antibody; there are several types of receptors, some (such as FcγRIV and FcγRIII) lead to effector response activation, whereas other receptors (such as FcγRIIB) cause inhibition. To better understand how subclasses regulate immune responses, Nimmerjan and Ravetch (2005) studied binding of IgG subclasses to different receptors. For their investigation, an Fab region that recognizes an antigen called TA99 was grafted onto Fc regions for the different IgG subclasses. In one experiment, the affinity of each Fc + TA99 for different receptors was measured. Since the presence of the carbohydrate fucose in the Fc oligosaccharide had been previously reported to influence receptor affinity, antibodies with and without fucose present were compared. Some of the results are presented in the table below. IgG subclass IgG1 (+fucose) IgG1 (- fucose) IgG2a (+ fucose) IgG2a (- fucose) IgG2b (+ fucose) IgG2b (- fucose) Binding affinity for receptors FcγRIV or FcγRIII FcγRIIB 0.3 3.3 0.5 1.3 29 0.4 310 3.3 17 2.2 200 10 A/I ratio _____ _____ _____ _____ _____ ______ A. Explain the nomenclature used in naming the receptors, such as “FcγRIV”. B. Calculate the ratio of activator receptor to inhibitor receptor affinity (A/I ratio) for each type of antibody. C. Which subclass(es) would appear to activate immune responses? Which would appear to inhibit response? Explain. D. What is the effect of fucose removal? Does it increase affinity for activating or inhibiting receptors? Explain. E. The results suggest two ways in which the immunological response to antigen+IgG can be regulated; explain. (Nimmerjah F, Ravetch JV.2005. Divergent immunoglobin G subclass activity through selective Fc receptor binding. Science 310:1510-1512.) Biol 430 Question Bank Antibodies Page 6 12. The properties of, and the effector responses to, different antibody isotypes is exquisitely adapted to its particular immunological role. In the table below, identify the isotype of the antibodies being described, and then explain how those properties relate to the immunological function. Type Properties Pentameric structure _____ Low affinity Activates complement Relation of Properties to Immunological Function Dimeric structure _____ Large quantities secreted Doesn’t activate complement High numbers in serum and lymph _____ Activates complement Triggers ADCC and opsonization Respond to eukaryotic antigens _____ Triggers Mast cell ADCC Associated with mucosyl tissues Some of these questions have been adapted from Goldsby et al (2003) Immunology, ed 5. Biol 430 Question Bank Antibodies Page 7