Antibody structure and AbAg interactions week 2 Flashcards
What are the 5 classes of antibodies?
What are the subclasses of antibodies?
- 5 Classes - IgG, IgM, IgD, IgA, IgE
- IgG exists in 4 SUBclasses:
- IgG1, IgG2, IgG3, IgG4 (numbered in order from most concentrated in plasma to least concentrated)
• IgA also has 2 subclasses, IgA1 and IgA2 (numbered in order from most concentrated in plasma to least concentrated)
How many chains do Abs (antibodies) consist of? How are the chains linked to one another?
What is valence?
• Molecules are SYMMETRICAL, consisting of a basic 4 polypeptide unit of 2 identical heavy chains and 2 identical light chains
- The chains are linked together by covalent disulfide bonds as well as by non-covalent bonds
• VALENCE = # of Antigen binding sites-how many antigens and antibody will bind
What are domains? How many domains are contained within heavy and light chains of Igs?
At what end of the protein (N or C terminal) are the variable domains located? Why are they called variable domains? Where are the constant domains located?
Each polypeptide chain contains domains, which are compact globular regions containing 100-110 amino acids and are formed by intra-chain disulfide bonds. Light chains contain two domains, and heavy chains contain four or five domains.
The N-terminal domain of each heavy and light chain displays more variation in amino acid sequence than the other domains. Accordingly, the N-terminal domain of each polypeptide chain is designated as the variable domain (or region), and the remaining domains are designated as constant domains (or regions). The constant regions exhibit little variation in amino acid sequence and have very similar secondary and tertiary structures.
– Light chain: 1 variable (VL) , 1 Constant (CL)
– Heavy Chain: 1 variable (VH), 3 or 4 constant (CH1), (CH2), (CH3)
What class of molecules are immunoglobulins?
glycoproteins
Where are most of the oligosaccharides of immunoglobulins attached?
All immunoglobulins are glycoproteins, but the amount of carbohydrate varies with the immunoglobulin. Most of the oligosacchrides are attached to the constant domains of the heavy chain.
What are theclasses of light chains? In what domains do the light chains differ structurally? Which of the light chains is more predominant in human Igs? How many different light chains can be in one Ig?
What are the classes of heavy chains? Where do they differ structurally?
There are two classes of light chains, kappa and lambda. Both are approximately 25 kD in size, but they differ structurally in the constant domain. Both types of light chains display an equal ability to associate with heavy chains, although the ratio of kappa to lambda light chains in human immunoglobulins is approximately 2:1. However, one immunoglobulin molecule contains two identical kappa or two identical lambda chains.
Heavy chains vary in size from approximately 50 kD to 75 kD and are divided into five classes: gamma; alpha; mu; delta; and epsilon. The five classes differ structurally in their constant domains, and it is the structure in the constant domain that determines the class of the antibody chain, as well as its biological function.
Where is the protease sensitive region within immunoglobulins located? What is this region called?
Immunoglobulins possess a protease-sensitive region between the first and second constant domains of the heavy chain (CH1 and CH2). This region is more exposed than other portions of the immunoglobulin molecule and is also more flexible. Hence, the region is termed the hinge region.
What are the products of papain acting in the hinge region of Igs? What are the products composed of? What are the functions of the products? How many antigens can the products of the enzyme bind?
What are the products of pepsin acting in the hinge region of Igs? What are the products composed of? What are the functions of the products? How many antigens can the products of the enzyme bind?
The enzyme papain acts in the hinge region to cleave the antibody molecule into two Fab (Fragment, antigen-binding) fragments and one Fc (Fragment, constant) fragment. Each Fab fragment is composed of a light chain and the variable and first constant domains of the heavy chain. Each Fab fragment contains one antigen-binding site. The Fc fragment is composed of the carboxy-terminal portion of the heavy chains and initiates biological activities subsequent to antigen binding.
A second enzyme, pepsin, acts at the hinge region to cleave the antibody molecule into one F(ab’)2 fragment and a degraded Fc fragment. Each F(ab’)2 contains two covalently-linked Fab pieces and consequently is bivalent (i.e., contains two antigen-binding sites).
pepsin cleavage forms a pepsi cup
Explain the ability of Fab and (Fab)2 to crosslink with other antibodies.
Antibodies can crosslink antigens and form large lattices that precipitate in solution. Because (Fab)2 has a valence of 2, it has the capacity to do this. Fab cannot crosslink antigens because they only have one binding site.
What is the most abundant Ig in serum?
What is the primary antibody formed in secondary immune responses?
What is the only Ab that can cross the placenta?
What is the most important activity of Abs?
IgG constitutes 75% of total serum immunoglobulin in adults. It is the major antibody formed during the secondary immune response. IgG is the only antibody that can cross the placenta. (side note: This is an active transport process, receptor mediated. newborn baby has adult levels of IgG from mother. provides immunity for first 3-6 months of life until infant can produce own IgG)
Opsonization is the most important activity of antibodies.
What are the functions of IgG molecules?
- Antigen Binding: antibodies can neutralize antigens/toxins just from binding
- Opsonization (bind particle to phagocyte)
- Activate complement
- Antibody-Dependent Cellular Cytoxicity (ADCC) (an effector mechanism of cell-mediated immunity). IgG is the only antibody with this function!
State the functions of the following domains of IgG molecules.
VH + VL
Cγ1
Cγ2
Cγ3
VH + VL = Antigen Binding
Cγ1 = None
Cγ2 = Complement Activation
(C1q binding)
Cγ3 = Fc receptor binding.
(Fcγ receptors are found on all phagocytic cells [neutrophils, eosinophils, monocytes, macrophages, NK cells, placental tissue, immature DCs).
Attached pic: Fcgamma receptor on phagocyte recognizes Fc region in the Cγ3 region of IgG molecules. Note that this process works synergistically with complement and increases phagocytosis 4k fold. Some bacteria are resistant to phagocytosis due to a slippery polysaccharide capsule. Opsonin overcomes this because phagocytes bind to Abs or complement molecules and use them to phagocytize bacteria.
Describe the difference in activity of the four different subclasses of IgG molecules.
- IgG exists in four subclasses, named in decreasing order of serum concentration: IgG1, IgG2, IgG3, and IgG4
- ALL subclasses are opsonins
- ALL subclasses cross the placenta
- ALL subclasses mediate ADCC
- ONLY IgG1, 2, and 3 activate complement; IgG4 does NOT
Note that IgG1 and IgG3 work best for the first 3 listed activities (but Ig2 and IgG4 also function in those activities)
What is the structure of IgM in serum? What is the valence of the form of IgM in serum?
What cell uses IgM as its antigen receptor? What form is IgM in on these cells?
IgM is a pentamer in plasma. Because it is a pentamer is serum, its valence is 10. Each of the five monomer subunits is identical and contains two (mu) heavy chains and two light chains. The heavy chain contains an additional domain; therefore, the size of the mu heavy chain is about 65 kD (110 amino acids) larger than the gamma heavy chain.
IgM is expressed on B lymphocytes, where it mediates antigen-specific B lymphocyte activation. On B lymphocyte, IgM is found as a monomer, and it is the antigen receptor for the B cell.
How are the monomer subunits linked in IgM to form a pentamer? What closes the pentamer? What kind of molecule is it?
What are functions of IgM?
The monomer subunits are linked by disulfide bonds between adjacent CH3 and CH4 domains, and the pentamer is closed by the J chain, a 15 kD glycoprotein that covalently links (via disulfide bonds) two monomer subunits together.
Functions:
- IgM is the major antibody class formed in the primary immune response
- Most efficient activator of complement
- Expressed on B lymphocytes where it mediates antigen-specific B-lymphocyte activation: triggers activation, proliferation, differentiation of B-cells
What is the predominant form of IgA in serum? What other form does it exist in in serum?
Where is IgA predominantly found? What form does it exist in in this location?
IgA is present in serum and is the predominant antibody in seromucous secretions. (external secretions: tears, sweat, saliva, in GI tract, respiratory tract, GU tract, breast milk, etc.)
Serum IgA constitutes 15-20% of serum immunoglobulin and exists largely (>80%) in a monomeric structure analogous to IgG. Serum IgA also forms dimers (20%).
Secretory IgA is present almost exclusively in seromucous secretions and exists primarily as a dimer.
The heavy chain is of the alpha class, and there are two subclasses of IgA: IgA1 and IgA2.
How are the monomers linked in IgA to form dimers? Is this a covalent or noncovalent bond? What is the valence of the dimer form?
What is secretory component? What IgA molecules have secrotory component? What secretes it? What are its functions?
The two monomers are linked covalently by a J chain (same as for IgM). Each secretory IgA molecule also contains a secretory component, which is a 70 kD polypeptide fragment of a receptor for polymeric immunoglobulin on the basal surface of mucosal epithelial cells. Secretory component is the only part of any Ab molecule not made by B lymphocytes or plasma cells–> synthesized by by epithelial cells.
The secretory component (or piece) facilitates transport of the secretory IgA across the mucosal epithelium into the lumen and protects the secretory IgA from proteolysis. There are a lot of proteases in the environment IgAs reside-example is digestive enzymes in saliva and GI tract. Secretory IgA is responsible for immunity at mucosal surfaces.
What are the functions of IgA?
- NO effector functions: when binds antigens does not trigger biological response. doesn’t activate complement, mediate phagocytosis, granule release from mast cells, etc.
- Binds Ag
– Neutralizes viruses
– Neutralizes toxins
– Blocks bacterial adherence (for example, in UTI, bacteria need to attach to colonize)
There is no biological response to IgA binding to antigens because if so, you would have an inflammatory response every time you touch something, take a breath, eat, etc.
What form is IgD found in? What is the predominant location of IgD? What is its function?
What form is IgE found in? What is the relative amount of IgE in serum? What is the function of IgE? What type of antigen does it protect against?
IgD is a monomer (delta heavy chain) of 184 kD. It is the predominant immunoglobulin, with IgM, on mature B lymphocytes, but comprises <1% of total serum immunoglobulin. Like IgM, IgD can mediate B cell activation.
IgE is a monomer (epsilon heavy chain) of 180 kD. The epsilon heavy chain contains an additional constant domain. IgE comprises <0.004% of total serum immunoglobulin, but binds strongly to Fc receptors for IgE on basophils and mast cells. IgE triggers granule release from mast cells and basophils, aiding in host defense against metazoan (worm) parasites. IgE also mediates immediate hypersensitivity reactions.
Allergies are anti-parasite responses against non-parasite Antigens.
What antibodies are present on the surface of memory B lymphocytes? What end of the chain (N or C) and which chain (heavy or light) is anchored into the membranes of B lymphocytes?
IgG, IgA, or IgE are present on the surface of memory B lymphocytes, where they serve as antigen receptors. The membrane-bound form of IgA is a monomer. The membrane form of the antibodies are anchored by a hydrophobic (lipophilic) segment at the carboxy-terminus of the heavy chain that arises from alternative RNA splicing of the heavy chain message.
What are isotypes? How many isotypes do humans have?
Isotypes= Ab Class & Subclass
• Defined ONLY by CONSTANT region
Isotype defines the structural differences in the constant regions that distinguish the class and subclasses of heavy chains and the class of light chains within a single species. Each isotype is encoded by a specific gene. Humans have 9 heavy chain isotypes (γ1, γ2, γ3, γ4, α1, α2, δ, ε, and μ) and 2 light chain classes (κ and λ).
What are allotypes? What is the inheritance pattern for allotypes?
Allotype defines small structural variations in the constant region of the heavy and light chains of an immunoglobulin of the same isotype within the same species. The genes encoding heavy chains (most notably those of IgG) and light chains are polymorphic. The structural variations encoded by the different alleles result from the substitution in many cases of only one or two amino acids. The number of allotypes for human IgG ranges from 0 for IgG4 to 19 for IgG3. Allotypes exhibit Mendelian form of inheritance.
Allotypes are biologically insignificant, may be used for paternity/maternity testing.
What are idiotypes?
Idiotype is determined by structural differences within the variable domains of heavy and light chains. Each clone of antibody-producing lymphocytes synthesizes an immunoglobulin molecule with a unique variable region amino acid sequence. This sequence dictates the antigen specificity of the antibody.
idiotype comes from idiosyncrasy which essentially means unique.
What are myeloma proteins? What is Bence-Jones protein? In what disease is it present? Where is it detected in patients with multiple myeloma?
Myeloma proteins are immunoglobulins synthesized by a single clone of a malignant plasma cell. Therefore, a myeloma protein is monoclonal (i.e. derived from a single clone) and has been observed with all immunoglobulin classes. Free light chains termed Bence-Jones proteins are found in the urine of patients with multiple myeloma.
What is immunogenicity? What is the difference btwn and antigen and an immunogen?
Adaptive immunity is the specific response of the immune system to foreign substances or molecules, which are termed antigens. Although the terms are often used interchangeably, strictly speaking, immunogenicity defines the capacity of a substance to react with and activate the immune system. Thus, immunogens are substances that induce an immune response, and antigens are substances that react specifically with the immune system. All immunogens are antigens, but not all antigens are immunogens.
Note: Antigens arise from both external and internal sources. Externally-derived antigens may be soluble molecules such as bacterial toxins, or components of more complex structures such as bacteria or viruses. Endogenously-derived antigens are expressed on the cell surface of virus-infected cells or tumor cells. Antigens may be: proteins (toxins, allergens, viral proteins, etc); polysaccharides (bacterial cell walls and ABO blood group antigens); lipids; organic molecules; inorganic molecules, and nucleoproteins.
What is the nature of Ab-Ag interaction? Is it covalent or non-covalent?
Antigen-Antibody Interactions are NEVER COVALENT
What are the three criteria for immunogenicity?
F. Criteria for immunogenicity
- Foreign. The immune system is set up to distinguish self from non-self. With limited exceptions, only foreign (non-self) molecules are immunogenic.
- Molecular size. A minimum size is necessary, but there is no specific size threshold. In general, molecules <5 kD are weakly immunogenic, if at all, whereas molecules >100 kD are strongly immunogenic.
- Chemical complexity. Immunogenicity increases with complexity. Homopolymers of a single amino acid are poor immunogens, whereas polymers of repeating units of 2 or 3 amino acids may be very immunogenic.
What are antigenic determinants/epitopes? What is the typical size of an epitope?
What are haptens? What are carriers?
Only limited portions of antigenic molecules interact with an antibody. These areas are termed antigenic determinants or epitopes. The number of distinct antigenic determinants varies with size and complexity of the molecule. However, a given individual may respond to only a subset of determinants. Epitopes that induce an antibody response in the majority of individuals are termed immunodominant epitopes.
Antigenic determinants are usually 5-7 amino acids for proteins or 5-7 monosaccharides or polysaccharides. Thus, they have similar molecular dimensions. In general, the antigenic determinants for antibody interactions are on the most exposed (hydrophilic) regions of a molecule.
Hapten - a single, isolated antigenic determinant. Haptens are small, chemically defined molecules that are not immunogenic but that can react with antibody of appropriate specificity. Thus, haptens are the one class of substances that are antigens but not immunogens. Haptens are always univalent molecules, (i.e., have a single epitope) in contrast to multivalent proteins or polysaccharides. Haptens (e.g., dinitrophenol, DNP) act as a partial or complete antigenic determinant when coupled to a protein carrier. The protein carrier has its own inherent antigenic determinants. Some antibodies made against the hapten-carrier complex will be specific for the hapten. Virtually any chemical structure may act as a hapten if coupled to a protein carrier.
What is a conformational determinant? What is a sequential determinant?
In what part of the molecule are sequential determinants typically located?
Denaturation will destory what type of determinant? (conformational or sequential)
Conformation and sequence are each important to the antigenicity of proteins and polysaccharides. Thus, a conformational determinant is based on the overall structure of a substance, e.g., globular versus helical. In contract, a specific sequence of amino acids or monosaccharides defines a sequential determinant. The sequences may be either terminal or internal, but the sequences are almost always localized in hydrophilic regions of the molecule.
Denaturation of the antigenic molecule destroys a conformational determinant, but not a sequential determinant. In some cases, denaturation may uncover a new sequential determinant that is not accessible in the native molecule.
What are hypervariable regions? Framework regions? What are the functions of each?
How many hypervariable regions to light and heavy chains have?
What is a complementarity determining region? Why is complementariy important in AbAg interactions?
As described previously, the antigen binding site of an antibody is formed by the variable domains of the heavy and light chains. The variability in the amino acid sequence in the variable domain, however, is not uniformly distributed throughout the domains. Regions that show the greatest variability are termed the hypervariable regions, whereas the relatively more constant sequences between the hypervariable sequences are termed the framework regions.
Light chains have three hypervariable regions, and heavy chains have three or four hypervariable regions. A specific variable domain may be linked to any constant domain of a heavy chain class.
The homologous domains (i.e., the variable or constant domains) of the heavy and light chains are paired in the Fab. More precisely, the variable domains of the heavy and light chains are aligned to bring together the hypervariable regions of each chain. It is the hypervariable regions of the heavy and light chains together that form the antigen binding site. The framework regions are important in the folding and proper orientation of the binding site.
The amino acids in the hypervariable regions interact with the amino acids or monosaccharides that comprise the antigenic determinants. Therefore, the molecular dimensions of the antigenic determinant and the antigen binding site must be complementary to each other. (For this reason, the hypervariable region is sometimes referred to as the complementarity-determining region.) The strong association between antigen and antibody is the result of multiple non-covalent bonds (e.g., hydrogen, Vander Waal, and electrostatic) formed between the antigen binding site and the antigenic determinant. Complementarity is necessary for a close fit, because the strength of the non-covalent bonds is inversely related to the distance between the interacting groups.
What is cross-reactivity? How strong is AbAg binding in cross-reactivity?
Studies using haptens have shown that antibodies react very specifically with the antigenic determinant that induced the antibody formation. However, antibodies made against one antigen or hapten may, in some cases, react with another antigen or hapten. This phenomenon is termed cross-reactivity and reflects shared antigenic determinants by the two antigens. Generally, an antibody binds less strongly to a cross-reacting antigen than to the antigen that induced the antibody formation.
Why is the AbAg complex essentially irreversible under normal conditions?
What is affinity? What is avidity?
- The reaction between an antibody and an antigen is reversible, but dissociation of the antibody-antigen complex is slow, such that the antibody-antigen complex is essentially irreversible under normal conditions.
- Affinity is a measure of the strength of the interaction between a single antigen epitope and a single antigen binding site (Fab) of an antibody. The interaction follows the Law of Mass Action, which allows the affinity to be measured experimentally. This calculation holds for a hapten (univalent interaction) and Fab, but provides only an approximation for a multivalent antigen.
- Avidity is a measure of the strength of the binding between a multivalent antibody and a multivalent antigen. IgG, serum IgA, IgD, and IgE each have two antigen binding sites, whereas secretory IgA and IgM have four and ten binding sites, respectively. Avidity is dictated by the affinities of the individual interactions, but the avidity is greater than simply the sum of the individual affinities.
What kind of AbAg interactions result in agglutination?
What kind of AbAg interactions result in precipitate? What is the formation of precipitate a function of?
Antigen-antibody complexes are also referred to as immune complexes. The form taken by the antigen-antibody complex depends on the nature of the antigen and the ratio of the antigen and antibody concentrations. Antibody interactions with particulate antigens (such as intact cells) results in agglutination of the cells, whereas binding of antibody to soluble antigens forms immune complexes that may precipitate. The formation of a precipitate is a function of the antigen and antibody concentrations.
How is precipitation of an immune complex observed? What are the 3 zones of precipitation that can be distinguished? List and describe these zones. State what kind of AbAg interaction occurs in each zone.
Precipitation of an immune complex can be observed when increasing amounts of an antigen are added to a series of test tubes, each containing a constant amount of antibody. As the amount of antigen added increases, the amount of precipitate increases and then decreases. Three zones of precipitation can be distinguished: antibody excess; equivalence; and antigen excess.
In antibody excess, the concentration of antibody is in excess relative to the concentration of antigen. Consequently, most of the antibody is bound in a univalent fashion to antigen.
In antigen excess, the concentration of antigen is in excess relative to that of the antibody. Ultimately, one antibody molecule will bind and cross-link two antigen molecules.
At equivalence, the relative concentrations of antigen and antibody are equivalent. Antibody such as IgG binds bivalently and ultimately can form an insoluble lattice with a multivalent antigen. An antigen must be at least bivalent for an insoluble lattice and, thus, a precipitate, to form. Other factors, such as number and heterogeneity of antigenic determinants, amount and heterogeneity of antibody, and antibody affinity, also influence lattice formation.
What is the clinical significance of immune complexes?
Similar changes in immune complex size are also observed clinically. In the initial stage following antigen exposure, antigen excess dominates until sufficient antibody is produced. The complexes remain soluble and are not cleared from the circulation. However, the complexes, due to their larger size, can become trapped in the tissues and eventually can cause tissue damage. As more antibody is produced following antigen exposure, equivalence and antibody excess are achieved. Phagocytic cells in the reticuloendothelial system clear these larger immune complexes from the blood via recognition of the Fc portions of the antibody molecules. The larger immune complexes can also activate complement and phagocytic cells in the circulation via the Fc region. Thus, in conditions where antigen exposure persists, such as in autoimmune diseases, formation of the immune complexes can overwhelm the reticuloendothelial system and lead to tissue damage.
