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63 Cards in this Set
- Front
- Back
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Q: When does immunization occur?
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-Immunization occurs when foreign or immunogenic material (containing the antigen) is introduced into a person or animal.
-The route of immunization must be such that the immunogen will persist for days (and not be rapidly cleared from the body). Dendritic cells or macrophages must find the antigen. |
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Q: What are good routes of immunization?
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-Subcutaneous or intramuscular injections are good routes of immunization.
-Antibodies, which are proteins that physically bind to the ‘native' antigen, are synthesized "on demand", and are detectable in the blood stream about 5 days after initial immunization. |
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Q: What happens after exposure to immunogens?
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-After intramuscular immunization, one class of antibody, immunoglobulin M (IgM) appears first, and then later, usually by 2 weeks after immunization, another class of antibody, immunoglobulin G (IgG), appears. At this time, the IgM antibody levels normally drop. An additional feature of immunization is that, while the concentration of IgG antibody in serum will decrease after several months, the affinity (binding strength) of the late response IgG antibody will be greater than the initial antibody. Antibody produced in response to booster immunizations will be mostly IgG (and not IgM) and also be of higher affinity
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Q: Describe the Ab levels before immunization.
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-Before immunization, there are usually no antibodies to the particular antigen. However, occasionally an animal or person may have already encountered a structurally related antigen (by prIor exposure) and will already have some "cross-reacting" antibodies. That is why "prebleeds" from the same people or animals are necessary for evaluation of vaccines or as controls for generation of antisera for diagnostic purposes.
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Q: What must happen to T cells for immunization to occur?
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-T (as well as B cells) must respond, T cells react to protein antigens. Since T cells can only recognize , foreign peptides (30 amino acids or less) presented in MHC molecules, the large 'foreign' proteins are proteolytically digested into smaller peptides and then bound to MHC proteins. The peptide-MHC complexes appear on the plasma membranes of antigen-presenting cells (APCs). Only when T cells react and produce growth factors can B cells make IgG, IgA or IgE antibodies.
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Q: Define antigen for B cells.
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-(Ag) a substance that will bind to an antibody protein. Antigens for B cells include proteins, as well as nonprotein substances such as carbohydrates, lipids and some chemicals.
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Q: Define antigens for T cells.
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-a peptide that is derived from a foreign protein and that will bind to MHC molecules and interact with T cell receptors (TCRs).
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Q: Define immunogen.
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-a substance with antigens that elicits an immune response in VIVO. The immunogen is what is actually given to the animal or person to stimulate antibody production or cellular immunity. Most immunogens contain both T cell antigens and B cell antigens., induce an immune response
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Q: Define antibodies (Abs).
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-(Abs) secreted proteins that will specifically bind to foreign substances. One antibody molecule will bind to one antigen or to a chemically similar antigen, but not to antigens with dissimilar structures.
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Q: Define immunoglobulins (Igs).
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-are the plasma glycoproteins that function as antibodies. All antibodies are immunoglobulins, e.g., IgG, IgM, IgA, IgD, or IgE.
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Q: Define Antiserum.
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-a serum which contains antibodies for the antigen in question, usually termed "antiserum to" a particular immunogen. Antisera (the plural) will always contain other immunoglobulins which are antibodies to antigens which the animal has encountered previously.
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Q: What is used to detect the presence of antibody?
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-Antibody proteins bind physically to antigens. A resulting antigen-antibody precipitate can often be used to detect the presence of antibody.
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Q: Define epitopes.
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-Epitopes are the particular chemical structures or regions that are recognized and physically bound by antibodies or by T cells. In the case of T cell receptors, the epitope includes a peptide from a foreign (nonselt) protein that is also bound by MHC molecules. may also be called antigenic determinants
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Q: Define haptens.
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-Haptens are small molecules that will bind to antibodies but are too small to induce an immune response by themselves (in free form). Haptens must be chemically coupled to macromolecular carrier proteins before they can become good immunogens. Optimal carrier proteins must be foreign (not self).
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Q: What properties make up a good immunogen?
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-The properties of a good immunogen to elicit antibodies are its "foreign-ness", size and charge.
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Q: Describe the foreign-ness property of a good immunogen.
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-Antigens must be foreign or "not self'. If a person or an animal has proteins of identical amino acid sequences the protein used as an immunogen, there I will be no antibody production. Human serum albumin from one person will be very similar in protein sequence, if not completely identical in amino acid sequence, to human serum albumin of another person and therefore will not be Immunogenic.
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Q: Is baboon serum albumin different enough from human serum albumin to be a good immunogen?
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-due to evolution, human serun albumin and baboon serum albumin will differ in amino acid sequence. A human being will make antibody to baboon serum albumin. Rat serum albumin will be even more different in sequence from human serum albumin than baboon serum albumin. Both will be immunogens to humans, but the rat albumin will be more immunogenic. Some proteins like viral proteins are completely foreign in amino acid sequence. A foreign protein will often have several regions that differ in amino acid sequence from the proteins of the potential responder.
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Q: What happens in terms of epitopes with different regions of difference?
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-Each region of difference will usually form unique epitopes. each unique epitope will be bound by different antibody molecules. The specificity of the immune response is exquisite
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Q: Describe the size propery of a good immunogen.
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-The sizes ofboth the epitope and the immunogen are important. The size of an epitope that will bind most tightly to antibody is about 6 sugars or 6 amino acids in length. This size is the largest structure that can fit into the antigen binding site of an antibody molecule.
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Q: What type of mechanism is involved in the binding of the epitope?
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-The binding of the epitope has been likened to a "lock and key" assembly, where the antigen is the key that fits into a complex site or groove that locks it into place. The epitopes are contained within much larger immunogens. A good immunogen is usually larger than 10,000 M.
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Q: Describe the charge property of a good immunogen.
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-Charged epitopes will confer more immunogenicity than uncharged epitopes to potential antigens. Charged amino acids are likely to be on the hydrophilic exterior of proteins where they are available to antibodies.
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Q: How was the difference between immunogenicity and antigenicity discovered?
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-The difference between immunogenicity and antigenicity was discovered by analysis of hapten-carrier immune responses. The key observation is that a hapten is an small antigen and will bind to antibody but that, in its free form (without covalent coupling to a protein), a hapten is not an immunogen Even though nonimmunogenic themselves, free haptens will bind to antibodies.
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Q: Describe the immune (Ab) response when a subject is given immunogen, hapten or both.
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-The injection of immunogenic carrier alone results in formation of carrier-specific antiuody. The injection of hapten alone does not result in formation of anti-hapten antibody. When the hapten is conjugated to an immunogenic carrier and injected, however, the animal responds by producing both anti-carrier and anti-hapten antibodies.
-in other words, Animals exposed to haptens covalently bound to foreign proteins make antibody to the haptens while animals given free haptens fail to make antibody to the haptens. |
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Q: Describe the experiment from above.
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-a hapten was chemically coupled to two different foreign proteins. The hapten (that was not immunogenic in free unbound state) became part of a larger immunogen when it was covalently coupled to a foreign protein. Now this complex immunogen can elicit antibodies to both the hapten and to the carrier protein. The foreign protein used to introduce the hapten is termed the carrier or conjugate protein. In synthetic "conjugate" vaccine synthetic peptide (containing the amino acid sequence of a pathogen) or carbohydrate structure is a hapten and is attached to a highly immunogenic carrier protein.
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Q: What are some examples of good immunogens?
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-Foreign (nonhuman) proteins are good immunogens. Charged chemical structures, like certain dyes, are good haptens.
-very large repeating units of sugars presented in membranes can sometimes be immunogenic, as can nucleic acids, when associated with immunogenic proteins. |
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Q: what are some examples of poor immunogens?
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-In general simple sugars, glycolipids, DNA and RNA are poor immunogens.
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Q: What does it mean for antibodies to cross-react?
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-bind to structures that are similar to the original Immunogen. When antibody is made to one epitope, it can cross react with structurally similar epitopes.
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Q: Give an example for Abs cross-reacting.
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-the chmicals were attached covalently to protein carriers through the amino group. The original hapten, attached to a carrier protein, was used to immunize a rabbit was meta-benzenesulfonate. The rabbit made anti-meta-benzenesulfonate antibodies which bind best to the original immunogenic epitope.
-When the sulfonate substituent on the benzene ring was moved to a different position (ortho or para) the hapten became different and some of the antibodies will cross react even though the rabbit was never exposed to ortho-benzenesulfonate or para-benzenesulfonate coupled to proteins. |
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Q: What happens in the experiment above if two changes were made to the hapten structure?
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-However, if two changes were made in the hapten structure (both the position and the chemical substituent) the anti-meta-benzenesulfonate antibodies did not bind. With the cross-reactive interactions, the antibodies bound with lower affinity than to the original epitope.
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Q: In the experiment above, can cross-reaction happen with Abs to proteins?
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-Similar cross-reactions can happen with antibodies to proteins. When the amino acid sequences of the original immunogen and another protein are very similar, antibodies can cross react. For example, antibodies to one influenza virus are likely to contain some antibodies that will react with a closely related but different strain of influenza virus.
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Q: What does the antigenicity of a protein epitope depend on?
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-The antigenicity of a protein epitope depends on the sequence of the amino acids and on the structure of the protein. Denature proteins can aquire new epitopes that are not found in native proteins and can also lose some native epitopes. Antigens must be exposed regions of proteins.
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Q: How many epitopes is an antibodies usually made to?
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-Antibodies are frequently made to only a few epitopes of an immunogen and not to the other epitopes, even though the other epitopes are "foreign" The sequence, conformation and location of a potential peptide epitope will affect whether or not it is recognized and is an immunodominant antigen.
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Q: What role do hydrophilic and hydrophobic sequences have on immunodominant regions?
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-Hydrophilic sequences usually generate immunodominant regions compared to hydrophobic sequences. Charged, hydrophilic regions of proteins interact with polar water molecules and will be exposed on the outside of a protein where antibodies, can bind to them. Hydrophobic regions of proteins are buried inside the folded protein and are not usually immunogenic.
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Q: How else can “foreign-ness” be made?
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-"Foreign--ness" can also result from generation of neoantigens recognized by When proteins are cleaved, the two new ends have amino and carboxyl charges. Even though the amino acid sequences are unaltered, the peptide is not the same and neoantigens may be produced. Also, when proteins are unfolded, the process can expose previously inaccessible determinants. in addition if proteins refold they can acquire new conformational determinants. Thus antibodies may recognize more determinants than were originally present on the immunogen.
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Q: Describe conformational, linear and neoantigenic determinants.
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-conformational determinant (discontinuous)-determinant lost by denaturation
-linear determinant-Ig binds to determinant in both native and denatured protein, Ig binds to determinant in denatured protein only -neoantigenic determinant-created by proteolysis, determinant near site of proteolysis |
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Q: Is it possible to have an instance where natural immunization is undesirable?
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-YES, allergies
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Q: What are the contents of vaccines?
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-There are vaccines which contain viruses (heat-killed or live-attenuated), purified proteins (e.g., toxoids or recombinant proteins), conjugate immunogens, or DNA that encodes protein immunogens.
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Q: How can live viruses be made less pathogenic?
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-Live viruses can be attenuated (made less pathogenic) by passage through cells or selective deletion of viral genes, and then used in vaccines. Toxoids are denatured protein toxins that have kept their antigens but lost their bioactivity.
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Q: What are the different types of vaccines?
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-In conjugate vaccines, the haptens contain the epitopes of the pathogens.
-For DNA vaccines against viruses, DNA encoding one or more viral proteins (but not the whole organism) is inserted in bacterial plasmids. The plasmids are grown in bacteria and then introduced to the body, taken into cells, where the inserted viral genes are expressed into protein. T cell responses and antibodies are made to the encoded viral protein |
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Q: What are synthetic vaccines?
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-Recombinant proteins, synthetic peptide-carrier immunogens and DNA vaccines are termed 'synthetic' vaccines because the antigens of the vaccine were not produced directly by the pathogenic organisms.
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Q: Are Abs affected by route of entry?
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-The route of entry affects the class of antibody made. Imrnunogens must cross into the body, entering across mucus membranes in the nose, lungs, gut, etc. or across skin barriers.
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Q: Define adjuvants.
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-Adjuvants are substances capable of potentiating and enhancing immune responses. They are mixed directly with the immunogen and administered to the animal. Adjuvants may serve to keep the immunogen from being cleared, as in the case of alum (alumium hydroxide) and oil adjuvants which are used for people. Many adjuvants promote inflammation. Adjuvants are often non immunogenic themselves.
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Q: Describe Freund’s complete adjuvant.
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-Freund's complete adjuvant contains oil and heat-killed Mycobacterium bovis, a tubercle bacillus that generates granulomatous walled-off lesions. These granulomas are useful because they provide a continuing source of antigen. Freund's incomplete adjuvant is used for boosters and lacks the bacilli. These adjuvants are now used only in animals.
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Q: Describe the structure of Ig.
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-each molecule of IgG has 4 protein chains (2 heavy (H) and 2 light (L)) and 2 antigen bindings sites, The four chains are held together by inter chain disulfide bonds of cysteine residues. Within a single immunoglobulin molecule, the 2 light chains are completely identical to each other and the 2 heavy chains are also completely identical to each other. One light chain and one heavy chain associate and are disulfide crosslinked to make a single antigen-binding site or "lock". These two antigen-binding sites are disulfide-linked through the heavy chains to make a complete immunoglobulin molecule with 2Ls, 2Hs and 2 antigen combining sites. Thus one IgG molecule can bind two antigenic epitopes and the epitopes must be identical or similar enough to fit into the same "locks". Intrachain disulfide bonds also form, making loops of the IgG protein that are distinct protein domains.
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Q: Describe the carboxytermini of the L and H chains.
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-The carboxytermini of the Land H chain proteins are aligned similarly. The most carboxyterminal domains form the constant regions.
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Q: Describe the amino termini.
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-The amino termini each have one variable domain. The variable domains of the L and H chains contain the antigen-binding regions. To allow antibodies to bind different antigens, the amino terminal domains of light and heavy chains of different antibodies vary in amino acid sequence.
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Q: Why is it important to have a variable sequence on the Igs?
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-The variable sequences are able to bind to various antigens and are called the variable (V) regions of antibodies. The domains that do not bind antigens are constant in amino acid sequence and called constant (C) regions. Light (L) chains have one variable and one constant domain. IgG heavy (H) chains have one variable and 3 constant domains.
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Q: What are the different forms of light chains?
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• The light chains of IgG can be either lambda or kappa light chains, which are encoded by different genetic loci. Since the light chains within a single IgG molecule are identical, they must both be either lambda or kappa.
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Q: What are the heavy chains of IgG called?
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-The heavy chains of IgG are called gamma heavy chains.
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Q: Describe the hypervariable regions of Ig.
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-Hypervariable regions have the most variation in the amino acid sequences within the variable regions of Land H chains and contribute to the parts of the immunoglobulin that physically interact with antigens. Light chains have 3 hypervariable regions. Heavy chains also have three hypervariable regions.
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Q: What is the complementarity determining regions (CDRs)?
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-Three Land 3 H, hypervariable regions are the complementarity determining regions (CDRs). The term "complementarity" is used because these are the regions that physically bind to the antigens
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Q: Describe the binding of antigens to antibodies.
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-antigen binding is not covalent
-The binding can be extremely strong, with affinity constants (Ka) as high as 10^11 M^-1. The affinity constant is the reciprocal of the concentration of free hapten at which half the antibody binding sites are occupied (the reciprocal of the dissociation constant, Kd). Even though the noncovalent binding is reversible, high affinity keeps antigens and antibodies bound together |
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Q: How many bindings sites does IgG Ab have?
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-Each IgG antibody molecule is divalent and will be able to bind two similar epitopes. These two epitopes may be on two molecules of antigen, or may be repeated epitopes on a single molecule of antigen.
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Q: What are some facts to remember in predicting antibody-antigen interactions?
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-One antiserum will contain many different antibody molecules that bind to many different epitopes.
-The two antigen-binding sites of each IgG antibody molecule are identical. -One immunogen or antigen can have repeats of the same epitope or have several different non-repeating epitopes. -Antibody-antigen dissociation is disfavored due to the high affinity of antibody. |
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Q: What happens with antigen-Ab interaction when in solution?
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-In solutions, antigen-antibody interactions can form a lattice containing many antibody molecules and many antigen molecules.
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Q: What determines the formation of the lattice?
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-Formation of the lattice depends on the valency of the antibodies (intact IgG has a valency of 2), the number of epitopes per molecule of antigen, and the ratios of antibodies to antigens. The number of epitopes per molecule of antigen is sometimes termed its antigenic valency
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Q: What does it mean when a protein is given an abbreviation (say BSA-DNP10)?
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-For example the abbreviation BSA-DNP10 designates the protein bovine serum albumin with 10 dinitrophenol (DNP) molecules attached to each molecule of albumin. Each DNP molecule is an epitope that can be bound by antibody.
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Q: What effect does Ab concentration have on complex size?
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-Small, soluble antigen-antibody complexes form in either antigen or antibody excess, whereas large lattice-like complexes form at antibody "equivalency". The large complexes usually fallout of solution (precipitate out from the supernatant). In (b) each of the dots of antigenic material contain several different epitopes and the antibodies are polyclonal, reacting to different epitopes.
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Q: What happens with the ag-ab complex get large?
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-When the lattice-like ag-ab complexes become sufficiently large, they precipitate out of solution and one can see the precipitate with the unaided eye.
-This concept of large v.s. small immune complexes is extremely important to understanding the pathogenesis of glomerular nephritis resulting from viral and bacterial infections. |
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Q: How would one be able to detect Ag-Ab complexes?
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-Antibody-antigen immunodiffusion, reactions in an agar gel allow for visual detection of specific antibody-antigen complexes and quantification of the amounts of antigen or antibody
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Q: What is the use of single radial immunodiffusions?
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-Single radial immunodiffusions can be used to quantify concentrations of antigen Antibody is mixed into liquid agar before it gels, wells are later cut in the gelled agar and antigen placed in the wells. The antigens near the well will diffuse because they are in antigen excess. The antigens will diffuse out in a halo until they are so dilute that they reach the precipitation point with the antibody in the agar. The more antigen, the further it must diffuse before getting to antigen-antibody equivalence. For wells containing the antigen, the bigger the ring, the greater amount of antigen initially placed in the well. Know how to read immunodiffusion plates.
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Q: What is the medical revelance of antigens, antigen-antibody and antigen-TCR interactions?
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-Antigenicity is important for recognition of pathogens, design of vaccines and understanding reactions like allergies and asthma.
-Conjugate vaccines are in use for several diseases, including the vaccine for bacterial pneumonia. The carrier protein for this vaccine is a recombinant, nontoxic variant of tetanus toxin and the haptens are Streptococcus pneumoniae bacterial carbohydrates. -Understanding antibody-antigen precipitation and solubility is essential to understand the logic and sensitivity of many diagnostic tests. -In vivo immune complexes contribute to the nephritis of lupus erythmatosus, to rheumatoid arthritis, and the immune complex nephritis that accompanies chronic viremic diseases |
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Q: What happens in the experiment above if other subsituents with negative charges, COOH- or arsenate were placed on the benzene ring and tested with the Abs?
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-the antibodies cross reacted if the substituents were kept in the same meta position, even though the rabbit was never exposed to meta-benzenecarboxylate or meta-benzenearsenate.
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