Antigen Structure and Antibody Interactions Flashcards
Immunogen
Generates either a humeral or cellular immune response
Antigen
Reacts with antigen receptors, irrespective of its ability to generate immune response: may or may not be immunogenic
Hapten
Small molecule able to react with preformed antibodies
Not immunogenic by itself, must be complexed to large protein (carrier)
Immunogenicity of antigens
Proteins > polysaccharides (B cells and Ab responses)
Proteins: T cell responses
Lipids and nucleic acids only immunogenic when complexed with proteins or polysaccharides
Properties of an antigen
Foreignness
Molecular size (>100kDa)
Chemical complexity and composition
Susceptibility to processing and presentation
Biological factors affecting immunogenicity
Genotype of recipient
Immunogen dosage
Rout of administration
Adjuvants
Adjuvants
Substances that when mixed with and injected with an antigen enhance the immunogenicity of that antigen
- Prolonged antigen persistence
- Enhance costimulatory signal
- Increase local inflammation, resulting in macrophage activation and antigen presentation
- Stimulate nonspecific proliferation of lymphocytes
Epitopes
Antigen receptors on lymphocytes recognize epitopes (antigenic determinants)
Usually immunologically active regions of an immunogen
Antigen recognition by B and T cells are fundamentally different and does not involve the same epitopes
B Cell Epitopes
Accessible and hydrophilic epitopes
Linear sequences of aa or non-sequential or conformation epitopes
Weak, non covalent interactions due to complementary shape between epitope and antigen-binding site
Can depend on shape of globular protein
Found on flexible region of immunogen
May have many different epitopes
T Cell Epitopes
Peptides complex with self MHC molecules on the surface of antigen presenting cells and other nucleated cells
Often internal and contain amphipathic peptide sequences revealed during processing
Hydrophobic residues interact with MHC molecules while hydrophilic regions interact with T cell receptor
Immunodominance is determined by ability to interact with MHC molecules of a given individual
Some T cells can recognize lipids and glycolipids presented by CD1
Mitogens
Capable of activating many clones of B cells or T cells, irrespective of their antigenic specificity and are known as polyclonal activators
Some prefer T or B cells
Superantigens
Activate large numbers of T helper cells by cross-linking their T cell receptors with any MHC2 molecule on an antigen presenting cells
Antigen-antibody interactions
Mediated by weak forces determined by close approach and multiple interactions
Exclusion of water molecules at site of interaction further increases the magnitude of weak atomic forces
NO COVALENT BONDS
Avidity
Overall strength of binding between a multivalent antibody and multivalent antigen
Cross-reactivity
antibodies in a polyclonal antibody preparation raised against an antigen can cross-react with a partial related antigen than bears one or more identical/similar epitopes
Precipitation reactions
Mixing an antigen in aqueous solution with specific antibodies (precipitins) in correct ratio resulting in formation of a visible precipitate
3D lattice structure formed by cross linking of multivalent soluble antigen with IgG or pentameric IgM
Can also be visualized in agarose gel
Radial immunodiffusion
Antibody incorporated in agar, antigen in center
Antigen diffuses out
Precipitate forms ring
Agglutination reaction
Cross-linking of particulate multivalent antigens by antibodies (agglutinins)
Antibody excess inhibits agglutination and is called prozone effect
Sufficient links must be formed to overcome mutual repulsion by like-electrical charged particles such RBC
IgM>IgG
Enzyme-Linked Immunosorbent Assay (ELISA)
Detects specific Ab (indirect ELISA) or antigen (sandwich ELISA)
Indirect ELISA
Antigen is coated to wells of a plastic plate in a monomolecular layer
Excess antigen is removed by washing and irrelevant protein is used to block remaining free sites on plastic
Patients serum is added
Enzyme-conjugated antiimmunoglobin (isotype) specific is added
Unbound antibody is removed by washing
Colorless substrate is added
Conor reaction generated by enzyme is quantified by absorbance using an automated plate reader
Sandwich ELISA
Capture Ab is coated to well before antigen containing preparation is added
Add second antibody then substrate
Measure colour
ELISPOT Assays
Modification of ELISA that allows measurement of molecules secreted by individual cells: known quantity of cells at bottom of cell with media
Wells are coated with detected antibody, washed and blocked
Cell population being studied is then added and cultured for a period of time to allow secretion of the molecule of interest
Cells are then washed away and detection antibody is added, followed by washing then substrate addition
Coloured precipitate giving spots
Can count spots and relate that to known quantity of cells
Immunofluorescence
Antigen-antibody complexes can be visualized under UV light if fluorescent dyes are added to Ab molecules
Direct: primary Ab bound to fluorochrome
Indirect: secondary Ab bound to fluorochrome (increase sensitivity, and primary antibody does not need to be coupled to fluorochrome)
Immunohistochemical staining
Staining of protein you are looking for
Counterstaining
Fix a section of tissue on a slide
Flow cytometry
Sample cells stained, in suspension
Pass through single file through laser
Forwards and side scattering of light from cells detected
Plotted on side/forward scatter and regions tell you what kind of cells are in solution
