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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
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
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
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
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
Describe the structure of the T cell receptor.
A Fab fragment immunoglobulin with alpha and beta instead of light and heavy
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
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
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
Where are peptides bound in MHC molecules?
The peptide binding cleft
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
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
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
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
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.
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
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
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
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
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
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
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
What is the role of adjuvants?
Adjuvants enhance the immunogenicity of substances mixed with it
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
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
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
How can affinity be measured?
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
What are monoclonal Abs?
- 1. Abs produced by a single clone of B lymphocyes
- 2. All the clones are identical
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