Antigen/Antibody Reactions (Bowden) Flashcards
Antibody
circulating antibodies
-soluble glycoproteins that recognize and bind antigens
-also function as membrane bound surface Antigen receptors on B cells and play a key role in B cell differentiation
they are the effector molecules of humoral immunity
5 classes of Antibodies
IgG IgM IgA IgE IgD
what makes up the class of an antibody?
The heavy chains
Variable regions
Fab (fragment antibody)
where antigen binding occurs
Constant regions
Fc (fragment constant regions)
biology activity (site of effector function)
CDR’s
Complementary-determining regions
aka Idiotope
aka hypervariable region
within variable regions of both H and L chains
(Show exceptional diversity)
Hypervariable regions that are involved in Ag binding by creating an interaction site that is complementary in shape, charge, hydrophobicity to the epitope it binds
6 CDR’s per antibody
Classes also known as …
isotype
Allotype
Allelic differences in the heavy chains
we all have IgG but we all have subtle differences in the IgG
Idiotype
Antigenic determinants on the V regions
we all may see an antigen, but we respond with slightly different V region determinants
see the antigen differently
called Idiotypic Network
Which Ig isotype is NOT bifunctional
IgD
usually only on surface of b cells
Antibody functions
Binds to antigen and then:
-promotes killing or removal of the immune complex
does this by:
- binding of the antibody to receptors expressed on host tissues
- binding of the antibody to the first component of the complement system to initiate the classical pathway
Immune complex
Antibody bound to an antigen
IgM
Pentamer- expressed on B cells
first antibody produced in primary response to antigen
good for binding Ag’s with multiple repeating epitopes (viruses, RBC’s)
good at binding complement
ha J piece
-binds to secretory cells- provides mucosal immunity
How long does it take for primary response to come up?
Two weeks
Why is IgM the first Ab made
Because IgM is the first constant region in the gene when looking at the constant regions
sIg
Secreted antibody
mIg
membrane bound antibody
IgG
80 percent of serum
functions:
- opsonization
- complement activation
- antibody dependent cell-mediated cytotoxicity
- neonatal immunity
- feedback inhibition of B cells
peaks at birth
4 Subtypes of IgG
IgG1
IgG2
IgG3
IgG4
All can cross the placenta
IgG1 and IgG3
Bind with high affinity to Fc receptors on phagocytic cells (antibody binds with its C region)
this is opsonization, which leads to increased phagocytosis
IgG2
restricted to carbohydrate Ag’s
binds with low affinity
IgG3
efficient activator of C’ b/c it has one more complement receptor
IgG4
binds with intermediate affinity
CD16
CD marker (Fc receptor for IgG)
located on NK cells, monocytes/macrophages and granulocytes
CD32
CD marker (Fc receptor of IgG) located on B cells (feedback inhibition), monocytes/macrophages and granulocytes
CD64
Fc Receptor for IgG
located on monocytes and macrophages
IgA
can have a J piece that binds secretory peptides (produced by epithelial cells)
primary antibody in external secretions (saliva, tears, mucus, bronchial, GU, digestive tracts)
Mucosal immunity***
Primary antibody for the entry point of antigens that you are exposed to through your mucosa
Polymeric-4 binding sites
Very important in baby immunity (breast milk)
IgE
Low conc. in serum, very short half life
Binds to basophils and tissue mast cells by Fc receptors with very high affinity
Asthma, hay fever, peanut allergies
Helminth infections
CD23 a and CD23b
Fc receptor for IgE located on mast cells and blood basophils
IgD
not usually in serum
usually in monomeric form present as an Antigen specific receptor on mature B cells
IgM and IgD on surface of B cells have what in common?
have same specificity
have the exact same complementary determining regions (CDR’s, aka idiotope, hypervariable region)
Antigens
foreign molecules that bind to an antibody or TCR ***whether or not they induce an immune response
Immunogens
Antigens that induce an immune response
Epitope
part of the Ag that contacts the Ag-binding sites of an Ab or TCR.
this is what binds the idiotope
aka antigenic determinate
Pathogen
organism that causes disease
Haptens
small molecular weight molecules that can bind to an antibody but must be attached to a large carrier macromolecule to stimulate an immune response specific for the small molecule
these are used in vaccination
also seen in allergies (penicillin)
Endogenous antigens
autoantigens- self antigen (autoimmune diseases)
alloantigens–> tissue specific antigen, present in one individual of a species but not in others (ABO)
Intracellular pathogens–> viruses, intracellular bacteria and parasites (chlamydia)
Exogenous antigens
enter the body or system and freely circulate in the body fluids and are trapped by APC’s
allergens–> immunogen
Microbial
Iatrogenic–> doctor induced
Factors influencing immunogenicity (whether something is immunogenic or not)
Molecular mass
Foreignness - usually only responds to non self
Chemical composition- more complex, more immunogenic
Physical form- particulate (more immunogenic) vs. soluble, denatured (more immunogenic) vs. native form
Degradability- Ags more easily phagocytosed are more immunogenic
Genetic factors- immunological repertoire
Age- very young and very old
Method of administration (dose, route, adjuvants)
Chemical nature of immunogens
Proteins- vast majority, good immunogens
Polysaccharides- good immunogens
Nucleic acids- poorly immunogenic, but can be immunogenic when complexed with proteins or single stranded (like viruses)
Lipids-non-immunogenic usually, but may be happens
what determines the size of the epitope?
The size of the antigen binding site on the Ab
Epitopes recognized by B cells
B cell interaction with antigens is highly dependent upon the 3-D conformation of the antigen (so don’t have to see things in a linear manner)
epitopes can be associated with both soluble or particulate immunogens
epitopes are usually exposed on the cell surface
Epitopes recognized by T cells
Only see primary sequence of amino acids in proteins (so not polysaccharides or nucleic acids)
so don’t need epitope on surface, b/c the T cell usually only see size 8-15 amino acid sequence on degraded smaller peptides
DO NOT recognize free peptides or soluble antigens
have to see peptides that are presented to them in bound to MHC (HLA) molecules
Epitope properties recognized by B cells
Accessible
hydrophilic
mobile peptides containing sequential or nonsequential amino acids
Epitope properties recognized by T cells
Internal linear peptides produced by processing of antigen and bound to MHC molecules
T dependent antigens
An Ag that requires both Th cells and B cells to stimulate an Ab response
always proteins***
required for class switching of antibodies and affinity maturation
T independent antigens
Non protein Ag’s (polysaccharides or lipids)
stimulate antibody response without T help
Usually identical epitopes that can cross-link BCR
Mitogens
Substances that cause cells (lymphocytes) to undergo cell division
LPS
a mitogen
activator of human B cells (does not need t cell help)
Superantigens
Antigens that activate a large fraction of the T cells
these are not processed but bind directly to MHC class II molecules and Vbeta of the TCR
diseases that happen from these are in part due to hyper-activation of the immune system and subsequent release of biologically active cytokines by activated T cells
Ab/Ag Binding
form non-covalent bonds (H bonds, electrostatic, Van der waals, hypdrophobic)
reversible interaction
Affinity
the strength with which one Ag-binding surface of an antibody (idiotope) binds to one epitope of an antigen
Avidity
Each antibody molecule can bind 2 to 10 epitopes of an antigen (depending if its IgA, IgM… etc.) or epitopes on two or more neighboring antigens.
The total strength of binding is much greater than the affinity of a single antigen-antibody bond
The avidity of an Ab for its Ag is dependent on…
the affinities of the individual Ag combing sites
how are hapten/carrier complexes used in drugs…
drugs alone are poor stimulators of immune responses due to simple/small structure
so…. these drugs form hapten-carrier complexes by binding a protein that is not immunogenic in free form
basis for many drug allergies
Monoclonal antibodies
antibodies derived from a single B cell clone
using this means that monoclonal antibodies against virtually any antigen can be produced
how do you make monoclonal antibodies
isolate spleen cells from mouse immunized with antigen X (Some of these are producing anti-X Ab)
fuse spleen cells with immortal myeloma cell line
Culture in medium
only fused cells (hybridomas grow)
select hybridomas that are producing monoclonal anti-X antibody
First drug for melanoma
Yervoy (Ipilimumab)
this is a monoclonal antibody that downregulates the immune response ???
ELISA
coat wells with antigen of interest
fill well with pt’s fluid of interest
if the pt’s fluid of interest contains antibodies against specific antigen it will bind
the wells are then washed…
next second antibody is added specifc for first antibody
also the second antibody has enzyme on it…
next wash again to remove all unbound second antibody
so next add substrate and if there is a color change then you know antibodies are present
Flow Cytometry
?
Recombinant antibodies
commonly used as therapeutic reagents in cancer immunotherapy
