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Draw and label the key components of an IgG molecule.
- 1. Three globular regions form a y shape
- 2. The two Ag-binding sites are at the tips of the arms which are held to the trunk by a flexible hinge region
- 3. They are made of 2 heavy chains and 2 light chains
- 4. The chains are joined by disulfide bonds
- 5. Each heavy chain is linked to a light chain and the 2 heavy chains are linked together
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Describe the structure of the Ig fold.
- 1. C-Domain - DEBA GFC
- 2. V-Domain - DEBA GFCC'C"
- 3. Disulfide bonds connect B and F
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Describe the difference between variable regions (framework, FR) and hypervariable regions (HV or CDRs).
- 1. There are 3 hypervariable regions designated HV1, HV2, and HV3 (CDR1, 2, 3)
- 2. HV3 is the most variable
- 3. The regions between the HV regions are framework regions, designated FR1, FR2, and FR3
- 4. The FR regions form the beta sheets that provide the structural framework of the domain
- 5. The HV sequences correspond to the 3 loops on the outer edge of the beta barrel away from the domain - the contact surface
- 6. The 3 loops from the Ig VH and VL domains come together to form the Ag binding site
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What are the noncovalent forces that hold together the Ag:Ab complex?
- 1. Electrostatic forces
- 2. Hydrogen Bonds
- 3. Van der Waals forces
- 4. Hydrophobic forces
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What are the different types of epitopes found on Ags?
- Continuous (linear)
- 1. Formed by a single small region of AA sequence
- 2. Abs that bind continuous epitopes can bind to the denatured protein
- 3. Detected by T cells
- Conformational (discontinuous)
- 1. Formed from several separate regions in the sequence of the protein brought together by protein folding
- 2. Abs that bind conformational epitopes bind only native folded proteins
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Describe the structure of the T cell receptor.
A Fab fragment immunoglobulin with alpha and beta instead of light and heavy
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What are the key differences between TCR Ag recognition and Ab Ag recognition?
- 1. Abs bind to the surface of protein Ags, binding discontinuous epitpopes
- 2. T cells bind to short continuous epitopes buried w/in the native structure of the Ag and must be exposed via fragmentation by APCs
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Describe the structure of the MHC class I molecule
- 1. 4 domains consisting of 3 alpha (ordered counterclockwise) and 1 beta2 microglobulin
- 2. The folded alpha1 and alpha2 domains form the walls of the peptide-binding cleft on the surface of the molecule
- 3. N terminus is at alpha2 and the C is at alpha3
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Describe the overall structure of the MHC class II molecule.
- 1. Same as MHC I
- 2. 4 domains consisting of 2 alpha, 2 beta subunits w/alpha on the left and beta on the left
- 3. The folded alpha1 and beta1 domains form the walls of the peptide-binding cleft on the surface of the molecule
- 4. N terminus is at alpha1 and the C is at alpha2
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Where are peptides bound in MHC molecules?
The peptide binding cleft
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How is the binding between MHC I and a peptide stabilized?
The amino and carboxy ends of the peptide contact the invariant sites at each end of the peptide binding cleft
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Describe the binding of peptides to MHC I through structurally related anchor residues.
- 1. The Ag peptides that can bind a given MHC variant have the same or similar AA residues at 2 or 3 particular positions along the sequence
- 2. These residues are called anchor residues
- 3. AA side chains at these positions insert into pockets in the MHC molecule
- 4. Most peptides that bind to MHC I variants have a hydrophobic anchor residue at the C-terminus
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What is the concept of anchor residues?
- 1. Specific residues in peptide fragments of Ags that determine peptide binding to MHC I molecules
- 2. Each MHC I molecule binds different patterns of anchor residues, called anchor motifs
- 3. This gives specificity to peptide binding
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Describe the binding of peptides to MHC II molecules.
- 1. Bound by peptide side chains that protrude into shallow and deep pockets of MHC cleft that are lined with polymorphic residues
- 2. Bound also by interactions between the peptide backbone and side chains of conserved AAs that line the cleft
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How do TCRs recognize peptides bound by MHC molecules?
- 1. TCR is aligned diagonally over the peptide and the peptide-binding cleft
- 2. The TCR-alpha chain lies over the alpha2 domain and the N-terminal end of the bound peptide
- 3. The TCR-beta chain lies over the alpha1 domain and the C-terminal end of the peptide
- 4. The CDR3 loops of both TCR-alpha and TCR-beta chains meet over the central AAs of the peptide
- 5. T cell receptor is threaded through a valley between the 2 high points on the 2 surrounding alpha helices that form the wall of the peptide-binding cleft.
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Describe the structure of the CD4 co-receptor.
- 1. Contains 4 Ig-like domains connected in chain
- 2. The N-terminal domain is designated D1 and is similar to an Ig V domain
- 3. The second domain is designated D2 or C2 and is not like the C or V domains
- 4. These two form a rod like structure and are linked to the other two by a flexible hinge
- 5. Peptide bound MHC II molecues bind the D1 domain
- 6. Recruits phagocytic cells for degradation
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Describe the structure of the CD8 co-receptor.
- 1. Heterodimer (alpha and beta chains) linked by a disulfide bond
- 2. It can exist as a homodimer of alpha chains
- 3. Alpha and beta resemble the Ig V domain
- 4. Carried by cytoxoic T cells that kill virus infected cells
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Describe the binding of CD4 and CD8 to MHC II and MHC I respectively.
- 1. They bind weakly to a V domain site in the alpha3 domain near the membrane and away from the cleft
- 2. They interact with residues in the base of the alpha2 domain
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Differentiate between the cellular expression of MHC I and MHC II.
- 1. MHC I - expressed in all nucleated cells and most highly in hematopoietic cells
- 2. MHC II - expressed on B cells, Mfs, DCs, and thymus epithelium
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What is the difference between an immunogen and an Ag?
- 1. Immunogen - any substance that on its own can elicit an immune response
- 2. Ag - any substance that can bind to an Ab
- 3. Some Ags need to be attached to an immunogen
- 4. All immunogens are antigenic but not all Ags are immunogenic
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What is the role of Ag dose in eliciting an immune response?
- 1. Very low doses do not elicit an immune response
- 2. Higher doses inhibit specific Ab production - low zone tolerance
- 3. Still higher doses produces a steady increase in response
- 4. Higher doses than this inhibit responsiveness - high-zone tolerance
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Describe several factors that influence the immunogenicity of proteins.
Larger size, intermediate dose, route (subQ vs. IV), complex composition, particulate/denatured form, different from self protein, slow release adjuvants, effective interaction with host MHC
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What is the role of adjuvants?
Adjuvants enhance the immunogenicity of substances mixed with it
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What is the difference between Incomplete Freund's, Complete Freunds, and alum?
- 1. Imcomplete Freund's - Oil-in-water emulsion; delayed release of antigen; enhanced uptake by Mfs
- 2. Complete Freund's - Oil-in-water emulsion w/dead mycobacteria; delayed release of antigen; enhanced uptake by Mfs induction of co-stimulators in Mfs
- 3. Alum - Aluminum hydroxide gel; delayed release of antigen; enhanced uptake by Mfs induction of co-stimulators
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How does affinity chromatography work?
- 1. Monoclonal Ab to Ag A bound to beads
- 2. Add mixture of molecules
- 3. Wash away unbound molecules
- 4. Elute bound molecules
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Define affinity and avidity
- 1. Affinity - the binding strength of a monovalent Ag to a single Ab binding site
- 2. Avidity - the binding strength of an Ab to intact Ag
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How can affinity be measured?
Eq dialysis
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Distinguish between isotypes, allotypes, and idiotypes.
- 1. isotypes - difference between C regions due to the usage of different c region genes
- 2. allotypes - differences due to different alleles of the same C gene
- 3. idiotypes - differences due to particular rearranged VH and VL genes
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What are monoclonal Abs?
- 1. Abs produced by a single clone of B lymphocyes
- 2. All the clones are identical
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How are monoclonal Abs made?
- 1. Spleen cells producing Ab from mouse immunized w/Ag A are mixed and fused with immortal myeloma cells lacking Ab secretion and HGPRT
- 2. The hybridomas are transfered to HAT medium where they proliferate and unfused spleen cells and myeloma cells die
- 3. Hybridoma that makes Ab specific for Ag A is selected for and cloned
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