Lecture 9: Antigen Structure & Interactions with Antibody Flashcards
what is the function of an immunogen?
generates either a humoral or cellular immune response
what is the function of an antigen?
reacts with antigen receptors whether or not it is able to create an immune response
Antigen binding to its receptor may or may not lead to an immune response, why might a response not happen?
certain small molecules react with preformed antibodies but are not capable of stimulating a specific immune response unless complexed with a larger protein
what are 4 types of antigens?
- proteins
- polysaccharides
- lipids
- nucleic acids
what type of antigens are the most potent?
proteins
what are the second most potent antigens?
polysaccharides
lipids and nucleic acids are not usually immunogenic unless ____
they are complexed with proteins or polysaccharides
proteins and polysaccharides activate ____ cells and induce ____ responses
B cells and immune presponses
proteins and glycolipids and lipids presented by CD1 induce ____ cell responses
T cell
what are 4 important properties of antigen?
- foreigness
- molecular size
- chemical complexity & composition
- susceptibility to processing and presentation
explain the “foreigness” property of antigens
antigens need to be recognized as non-self to create an immune response
antigens with a molecular size greater than ___kDa are optimal for immunogenicity and those less than ___kDa are generally poor antigens
100; 5-10
explain the role of chemical complexity and composition of antigens
the more complex an antigen, the greater its immunogenicity
describe the difference in immunogenicity between homopolymers and copolymers
homopolymers, regardless of their size, tend to lack immunogenicity while copolymers of sufficient size are immunogenic
what type of antigens would not be susceptible to processing and presentation to stimulate T cells (making them poorly immunogenic)?
macromolecules
what are 4 biological factors affecting immunogenicity?
- genotype of recipient
- immunogen dosage
- route of administration
- adjuvants
how does the genotype of the recipient affects immunogenicity?
some people may fail to mount an effective immune response to certain antigens while others may mount a more intense response because of their gene expression profile
how can immunogen dosage affect immunogenicity?
too much or too little antigen will fail to induce an immune response (insufficient activation or tolerance); repeated doses of immunogen will increase the strength of the immune response
how does route of administration affect immunogenicity?
intradermal and SQ are superior to the oral route
how do adjuvants affect immunogenicity?
they are substances that when mixed with antigen, increase their immunogenicity
what are the 4 ways adjuvants can increase the immunogenicity of antigen?
- prolong antigen persistence
- enhance the costimulatory signal
- increase local inflammation, resulting in macrophage activation and antigen presentation
- stimulate the nonspecific proliferation of lymphocytes
what are epitopes?
immunologically active regions of an immunogen
antigen receptors on lymphocytes recognize discrete sites on antigens called ____ or ____
epitopes or antigenic determinants
does the antigen recognition of B and T cells involve the same epitopes?
no; they are fundamentally different
in B cells, antibodies recognize native antigens by binding to epitopes that are ___ and ____
accessible and hydrophobic
in B cells, the antibodies may interact with ___sequences of AA or with ____ or ___ type epitopes
linear; nonsequential or conformational
how do antigens and antibodies bind to each other in B cells?
by weal noncovalent interactions due to complementarity in shape between epitope and antigen-binding site of the antibody
in B cells, most antibodies raised against a globular protein recognize surface structures that depend on the ____ of the native protein
conformation
B cell epitopes tend to be found in the ____ regions of an immunogen
flexible
in B cells; a large & complex protein antigen has many possible epitopes, but the _____ are the regions that are most accessible to antibody
immunodominant antigenic determinants
a ____ (large or small) antigen is more likely to bear a number of structurally distinct antigenic determinants
large
T cells only recognize peptides complexed with _____ on the surface of APC and other nucleated cells
self MHC
where are T cell epitopes typically located?
internally
T cell epitopes typically contain ____peptide sequences revealed by antigen processing
amphipathic
in T cell epitopes, the hydrophobic sequnces interact with ____ and hydrophilic sequences interact with ___
MHC; T cell receptor
what determines immunodominance of T cell epitopes?
the ability to interact with the MHC of a given individual
certain T cells can also recognize ____ and ___ presented by CD1 as antigen
lipids and glycolipids
what is the function of mitogens?
capable of activating many clones of B or T cells, irrespective of their antigenic specificity
mitogens are known as ___ activator s
polyclonal
what mitogens preferentially activate T cells?
lectin concanavalin A
what mitogens preferentially activate B cells?
LPS
what is an example of a superantigen?
staphylococcal enterotoxins
what is the function of superantigens?
activate a large number of T helper cells by cross-linking their T cell receptors with any MHC class 2 of an APC
superantigens are extremely potent ___
T cell mitogens
what type of bonding is involved in antigen-antibody reactions (4 types of noncovalent)
- ionic or electrostatic
- hydrogen
- hydrophobic
- Van der Waals
exclusion of water molecules at the site of antigen-antibody interaction ____ (increases/decreases) the magnitude of the weal atomic forces
increases
do the antigen-antibody reactions ever involve covalent binding? What is the significance of this?
no; this means their bonds are readily reversible
what is affinity?
the strength of the sum total of noncovalent interactions between a single epitope and an antigen-binding site of an antibody
what is avidity?
the overall strength of binding between a multivalent antibody and a multivalent antigen
when does cross-reactivity happen?
when antibodies in a polyclonal antibody preparation raised against one antigen cross-react with a partially related antigen that has one or more identical or similar epitopes
what are precipitation reactions?
mixing an antigen in aqueous solution with specific antibodies in the correct ratio results in the formation of visible precipitates
the antibodies involved in precipitation reactions are called ____
precipitins
what is the structure of the precipitates from the precipitation reaction?
3D lattice structure
how are the precipitates formed in precipitation reactions?
by cross-linking of multivalent soluble antigen with divalent (IgG) or pentameric (IgM) antibody molecules
t/f no precipitates are formed in there is an excess of antibody and or antigen
t
t/f precipitation reactions can be visualized in semi-solid media such as agarose gel
t
what are agglutination reactions?
cross-linking of particulate multivalent antigens such as cells by antibodies that are at least divalent leads to agglutination of particles
the antibodies involved in agglutination reactions are called ___
agglutinins
antibody excess that inhibits agglutination is called the ___ effect
prozone
during agglutination, sufficient links must be formed at the greatest possible distance to overcome ____ by particles with the same charge, such as RBC
mutual repulsion
which is a better agglutinin and why: IgM or IgG?
IgM because it spreads antigen the greatest possible distnace to overcome repulsion due to pentameric structure
what can be detected by Enzyme-linked Immunosorbant Assay (ELISA)?
specific antibody (indirect ELISA) or antigen (sandwich ELISA)
explain how an indirect ELISA works
- antigen is absorbed to teh wells of a plastic plate in a monomolecular layer
- excess antigen is removed by washing & irrelevant protein is used to block remaining free sites on the plastic
- excess blocking protein is removed by washing and the patient’s serum is added to the well
- after another wash, the enzyme-conjugated anti-immunoglobulin (isotype-specific) is added
- unbound antibody is removed by washing and the colourless substrate is added
- the colour recation generated by the enzyme is quantified by absorbance using an automated plate reader
in sandwich ELISA, a ____ is absorbed to the well before the antigen-containing preparation is added
capture antibody
what is a ELISPOT assay?
a modification of ELISA that allows measurement of molecules secreted by individual cells
how is an ELISPOT assay performed?
1wells are coated with detection antibody
- washed, and blocked
- cell population added and cultured to allow secretion of molecule of interest
- cells washed away
- detection antibody added
- washing & substrate addition
what is the purpose of Immunofluorescence reactions?
antigen-antibody complexes can be visualized under UV light if fluorescent dyes are first coupled to the antibody molecules
what is immunofluorescence used to assess?
antigen distribution on cells or tissues
what is the direct method of immunofluorescence?
the primary antibody is tagged with the fluorochrome
what is the indirect method for immunofluorescence?
- unlabelled primary antibody bound to the substrate is visualized with a fluorochrome-conjugated anti-immunoglobulin preparation that is specific for the isotype of the primary antibody
- Alternatively: use fluorochrome-labeled protein A, which binds the Fc region of certain immunoglobulin subclasses
what are 2 advantages of the indirect method for immunofluorescence?
- increased sensitivity bc several fluorescent secondary antibodies bind to each primary antibody
- simplicity bc each primary antibody does not need to be coupled to a fluorochrome
in flow cytometry using fluorochrome-labeled antibodies, the forward & side scatter of light gives an indication of cell ____ and ____ respectively and this is used for gating
forward: cell size
side: complexity
what shape antigens can bind to B cells? T cell?
B cells: 3D
T cells: linear complexed with MHC
NKT cells have special receptor that can recognize ___ complexes presenting lipids
CD1
TCR have ___ and ___ chains and signal through associated ____ chains
alpha and beta; CD3
what is the co-receptor for MHC found on helper T cells? What class of MHC does it bind to?
CD4 –> MHC2
what is the coreceptor for MHC on cytotoxic T cells? What class of MHC does it bind to?
CD8; MHC1
t/f T cell epitope are always sequential
t
mitogens and superantigens bypass the ____ of B and T cells
antigen specificity
superantigens are usually found in ___
microbes
what antibody has high avidity?
IgM (5x the binding sites = 5x the avidity)
what does it mean if there is a precipitation when an antigen is added?
you know the system has seen the antibody before bc it has antibodies that bind to it to form precipitates
ELISPOT is a more intense form of ELISA that can recognize a specific secreted ___
cytokine
