Antigen Structure and Antibody interactions Flashcards
What is an immunogen?
An antigen that can generate a humoral or cellular immune response
What is an antigen?
reacts with antigen receptors irrespective of its ability to generate an immune response or not
-may or may not be an immunogen
All immunogens are….
antigenic but not all antigens are immunogenic
what is a Hapten? when is it immunogenic?
Small molecule able to react with preformed antibodies
- is antigenic but can stimulate a specific immune response itself
- only becomes immunogenic when coupled to a large protein called a carrier
What is the most potent form of immunogens?
proteins
-can interact with both B and T cells
Which macromolecule ranks second in immunogenicity?
Polysaccharides
-can interact with B cells
Lipids and nucleic acids are not usually immunogenic unless?
They are complexed with proteins or polysaccharides
-not acting as haptens
What are the 4 properties of an antigen?
- Foreign: recognized as non self
- Molecular size: must be greater than 100 kDa to be an optimal immunogen
- Chemical complexity and composition: the more complex an antigen is, the greater its immunogenicity
- Susceptibility to processing and presentation: macromolecules that can’t stimulate T helper cells are poorly immunogenic
T cells can only recognize ____ structure?
Primary
B cells can recognize?
higher structures and conformation (2º-4º)
3 biological factors that affect immunogenicity (list)
- Genotype of recipient
- Immunogen dosage
- Route of administration
how does the genotype of the recipient affect the immunogenicity of the antigen?
some individuals may fail to mount an effective immune response to certain antigens while others may mount a more vigorous immune response as a result of their genetic composition
How does the dosage of immunogen affect its immunogenicity?
too much or too little antigen will fail to induce an immune response
What is tolerance?
a nonresponsive state due to exposure to high and low dosage extremes
How does the route of administration affect the immunogenicity?
IV and subcutaneous administration are superior to the oral route
Why is the oral route less effective?
exposes GALT to the immunogen BUT this route is specialized to be unresponsive
-the gut sees lots of foreign proteins so it’s trained to be less responsive
What is an adjuvant?
A substance that, when mixed with an antigen and injected will enhance the immunogenicity of that antigen
What are the 4 methods of action through which an adjuvant can work?
- Prolong the persistence of the antigen
- Enhance the costimulatory signal (increased response from sentinel cells)
- Increase local inflammation
- macrophage activation and antigen presentation - Stimulate nonspecific proliferation of lymphocytes
How are adjuvants different than carrier proteins?
there is no physical interaction between the adjuvant and the antigen
another term for antigenic determinant is?
epitope
What are epitopes?
Immunologically active regions of an immunogen
What is generally different between antigen recognition in B cells and T cells?
B cells recognize shape where T cells recognize primary structure
Binding of soluble antibody: T cell vs. B cell
B cells can bind soluble antigen where T cells cannot
What does the B cell interaction with antibody involve?
Binary complex of membrane Ig and Ag
What does the T cell interaction with antibody involve?
Ternary complex of T cell receptor, Ag, and MHC molecule
Epitope properties: B cells
B cells: can bind accessible, hydrophilic, mobile peptides containing sequential or nonsequential amino acids
Epitope properties: T cells
T cells: bind internal linear peptides produces by processing of antigen and presentation by MHC
What interactions occur between antibodies and antigens?
Weak noncovalent interactions due to complementarity in shape
Where do B cell epitopes tend to be found on an immunogen?
In the flexible regions
Where are the most immunogenic epitopes on an immunogen?
The hydrophilic ones on the outside
What is an immunodominant antigenic determinant?
Regions of the antigen that are the most accessible to antibody molecules
T cell epitopes often contain…?
Amphipathic peptide sequences and are internal
-sequences revealed by antigen processing
_____ residues interact with MHC while the _____ residues interact with the T cell receptor
Hydrophobic (MHC), Hydrophilic (T cell receptor)
How is immunodominance determined for T cell epitopes?
Determined by the ability to interact with MHC molecules of a given individual
Certain T cells can also recognize lipid and glycolipids presented by …?
CD1
What do mitogens do?
Activate many clones of B cells or T cells irrespective of their antigenic specificity
-called polyclonal activators
What mitogen preferentially activates T cells?
lectin concanavalin A
What mitogen preferentially activates B cells
LPS
What are superantigens (give an example)
Extremely potent T cell mitogens
-like staphylococcal enterotoxins
What is the effect of the superantigens?
Activate large numbers of T helper cells by cross-linking their T cell receptors with any MHC class II molecule on an antigen presenting cell
How does the superantigen exert its effects
Binds to the variable ß regions on the T helper cell
can be membrane bound or just interact with the antigen presenting complex at the MHC
Tricks the T helper cell into thinking it has the right antigen even if it doesnt
What 4 non covalent interactions contribute to the antigen antibody interactions?
- Ionic or electrostatic interactions
- Hydrogen bonding
- Hydrophobic interactions
- Van der Waals interactions
What makes antigen antibody interactions readily reversible?
Because they dont form covalent chemical bonds
Exclusion of ____ from the site of interaction further increases the magnitude of the weak atomic forces
water
What is affinity ?
the strength of the sum total of noncovalent interactions between a single epitope and an antigen binding site of an antibody molecule
What is avidity?
Overall strength of binding between a multivalent antibody and a multivalent antigen
What is cross-reactivity
antibodies in a polyclonal antibody preparation raised against an antigen can cross react with a partially related antigen that bears one or more identical or similar epitopes
An antibody with more antigen binding sites will have higher …?
avidity
what are precipitins?
visible precipitated antibodies resulting from the mixing of antigen in aqueous solution with specific antibodies
How are precipitins formed?
form a 3D lattice structure by the cross-linking of multivalent soluble antigen with divalent IgG or pentameric IgM
Precipitins are not formed under conditions of?
Antibody or antigen excess
- too much antigen: all the binding sites taken and not enough antibody to cross link
- too much antibody: only partial occupancy of the binding sites and no crosslinking
what is radial immunodiffusion?
Using a semi solid media like agar with antibodies on it you plate the antigen in the centre
The antigen diffuses out and complexes with the antibody and the diameter of the precipitate ring can be used to determine the concentration of antigen
What is an agglutination reaction?
Cross-linking of particulate multivalent antigens such as cells by antibodies that are at least divalent leads to agglutination of the particles
What is the prozone effect?
excess of antibody inhibits agglutination reactions from occurring
Why is IgM a more effective agglutinin than IgG?
sufficient links must be formed to overcome mutual repulsion by like-electrically charged particles such as red blood cells
-charged antigen particles will be held further apart
Which form of ELISA detects specific antibody?
Indirect ELISA
Which form of ELISA detects an antigen?
Sandwich ELISA
Step of Indirect ELISA
- Antigen coated to the wells of a plastic plate in a monomolecular layer
- Wash to remove excess antigen
- Blocking done using an irreverent protein in order to fill the remaining free sites on the well
- Wash again
- Substance to be tested is added to the well
- Wash again
- Enzyme conjugated anti-immunoglobulin is added
- Wash to remove unbound antibody
- colourless substrate is added to react with the enzyme
- colour reaction quantified by absorbance using an automatic plate reader
What is sandwich ELISA used for?
Measure antigens, cytokine levels, chemokines… mainly soluble factors
Steps in Sandwich ELISA
- Coast well with antibody
- Wash +block
- Add antigen to be measured
- Wash
- Add enzyme conjugated secondary antibody
- Wash
- Add substrate and measure colour
What are ELISPOT assays?
modification of ELISA that allows for measurement of molecules secreted by individual cells
-what proportion of cells are producing a certain molecule
ELISPOT assay procedure
- Wells are coated with detection (capture) antibody
- Wash + block
- Cell population being studied is then added and cultured to allow them to secret the molecule
- Cells are washed away
- Detection antibody added (binds to a different epitope than the capture antibody)
- Wash and substrate addition
How does Immunofluorescence work?
Antigen-antibody complexes can be visualized under UV light if fluorescent dyes are coupled first to the antibody molecules
When is immunofluorescence used?
to assess antigen distribution on cells of tissues
what is direct immunofluorescence?
the primary antibody is the one tagged with the fluorochrome
What is indirect immunofluorescence?
A primary antibody is bound to the substrate but is unlabelled
A secondary antibody that is specific for the isotype of the primary antibody and conjugated to a fluorochrome is then added
What are 2 benefits to the indirect immunofluorescence method?
- Increased sensitivity
- several secondary antibodies can bind to a single primary antibody - Simplicity because each primary antibody does not need to be coupled to a fluorochrome
What is immunohistochemical staining similar to?
similar to the ELISPOT. You fix cells or tissue against slide, wash with an CD4 specific antigen coupled to HRP
-then use a blue counter stain
How does flow cytometry work?
Sample of cells pass through in a single file
Laser light source shone at them will cause any cells labelled with fluorochrome to light up
-computer picks this up as a positive test
You can have more advanced machines that will actually sort the cells into + and - cultures for you
