L3: Antibodies and Antigens Flashcards
How are antibodies bifunctional molecules?
One end interacts w/ foreign antigen (can interact w/ at least 2 epitopes)
Other end interacts w/ components of host defenses (Fc receptors and complement proteins)
Where are antibodies found?
Serum and tissue fluids
Bound (noncovalently) to Fc receptors on the surface of many cell types
Integral membrane protein on the surface of unstimulated B cells (the only place that antibodies exist as integral membrane proteins)
What happens when an antigen binds to the B cell?
It differentiates and matures and becomes a plasma cell, meaning it is an antibody factory
What are antibody effector functions?
Opsonization: coating on bacteria which makes it easier to digest by phagocytes
Activation of complement
Antibody-dependent cell-mediated toxicity (ADCC)
Ab transport through epithelium or placenta: this is how passive immune protection is passed from mother to fetus
Activation of mast cells, eosinophils and basophils by IgE: these are involved in hypersensitivity reactions, asthma, and allergies
What is the basic structure of all antibodies?
2 identical light chains and 2 identical heavy chains
Light chains have one variable domain and one constant domain
Heavy chains have one variable domain and either 3 or 4 constant domains (depending on class of antibody)
What are the types of light and heavy chains of antibodies?
Light: lambda or kappa (about a 50/50 mix)
Heavy: alpha, delta, gamma, epsilon, or mu (each type is specific for an antibody class such as IgG, IgM, etc.)
How are the light chains held to the heavy chains in antibodies?
Disulfide bonds
How are the 2 heavy chains of antibodies held together?
Disulfide bonds
How are the individual immunoglobulin domains (within the chains) held together in antibodies?
Disulfide bonds
Where are there carbohydrates in antibodies? What do they do?
In the second constant domain
They help w/ solubility and protection from proteolytic digestion
What are hinge regions of antibodies susceptible to?
Being cut by bacterial protealases
What is the structure of immunoglobulin domains?
Antiparallel beta sheet structure
Why is beta sheet structure of immunoglobulin domains favored?
Although the beta sheet is limited (bc have to use small aa’s in beta sheet), the loops can have almost any aa’s bc the R groups are sticking out into the environment
Where is there the most variability in antibody structure?
In the complimentary determining region of variable domain of the light chain
This is where antigen interacts
What do the hinge regions of immunoglobulin do?
They impart flexibility and allow both arms of the antibody to interact w/ an epitope
Important for allowing antibody to bind and stay there for a long time
Epitope
The region of the antigen where the antibody binds
Paratope
Surface of the antibody where the epitope binds
What do secreted immunoglobulins bind to?
Bind to Fc receptors on the surfaces of many cell types; this is non-covalent binding
Antigen receptors
Immunoglobulins that are integral membrane proteins present on unstimulated B cells
What does binding of antigen to immunoglobulins on unstimulated B cell cause?
Activates the B cell and causes them to proliferate AND to produce the secreted form of the antibody that they expressed on their cell membranes?
How are membrane immunoglobulins locked into the membrane?
They have a hydrophobic transmembrane helix w/ polar groups at each end
What is the difference b/w membrane and secreted immunoglobulins?
These forms differ only at the C terminus
What causes the difference b/w the membrane and secreted immunoglobulins?
Difference is caused by changes in mRNA splicing (resulting in the use of a different polyadenylation site)
When immunoglobulin gets secreted, the entire transmembrane helix region gets replaced w/ a very small glycine-leucine-COOH
mRNA is transcribed and in RNA processing, there are 2 places where PolyA tail can be put on; if B cell is activated, polyadenylation factor gets upregulated and it will bind and direct the mRNA to use an earlier polyadenylation signal, which cuts off the transmembrane helix
What are the 5 immunoglobulin classes in humans?
IgG - gamma heavy chain IgM - mu heavy chain IgA - alpha heavy chain IgE - epsilon heavy chain IgD - delta heavy chain
How many immunoglobulin subclasses are there?
IgG, most common, has 4 subclasses
IgA has 2 subclasses
All others have 1
Characteristics of IgG
Major human serum immunoglobulin (60 - 70% of total circulating Ig)
Principal antibody mediator of the secondary immune response
Only antibody class that can cross the placenta and provide passive protection to the fetus
Primary antibody class used in research, diagnostics, and therapeutics
How do the 4 IgG subclasses differ?
Are 90 - 95% homologous in DNA sequence
Only real difference b/w them is the length of the hinge region and the numer and position of the interchain S-S bonds
Why is IgG3 particularly flexible and good at fixing complement? What is the disadvantage of this?
Has a long hinge region, so there is lower chance of steric hindrance when binding to antigen on one end and then binding to Fc or complement receptors on other end
Disadvantage is that teh long hinge region is also good for proteases and will get clipped
What are the relative serum concentrations of the IgG subclasses?
IgG1 > IgG2 > IgG3 > IgG4
Normal adult serum concentration of IgG
12.5 +/- 3 mg/ml
What are abnormal IgG serum concentrations? What can they indicate?
Below 0.6 mg/ml - immunodeficiency, monoclonal gammopathy of IgA or IgM
Above 17 mg/ml - Infections, liver diseases, autoimmune diseases, cystic fibrosis, sarcoidosis, polycythemia cera, 3rd trimester of pregnancy, myeloma, monoclonal gammopathy
Above 50 mg/ml - myelom, monoclonal gammopathy; is cancer or pre-cancer at these high ranges
Characteristics of IgM
10% of total antibody pool, almost all in circulation
Primary antibody mediator of the primary immune response
Agglutinates antigen - increases antigen visibility
Proficient at complement fixation
Structure of IgM
Pentamer
Heavy chains have C terminal tailpieces that make S-S bonds w/ the J chain
J chain is a cysteine-rich polypeptide that helps in antibody polymerization and secretion
Why is IgM the antibody mediator of the primary immune response?
When you see antigen for the first time, it is bound by IgM bc it is a pentamer and when it binds it doesn’t let go and summons the rest of the immune system cells to look at invader
Normal adult serum concentrations of IgM
1.25 +/- 0.5 mg/ml
What are abnormal IgM serum concentrations? What can they indicate?
Below 0.4 mg/ml - immunodeficiency, monoclonal gammopathy of IgA or IgG; think pre-cancerous or cancerous state that is raising the secretioni of the other 3 Ig’s, so there is suppression of IgM
Above 3 mg/ml - IgM monoclonal gammopathy, infection, parasitic diseases, liver diseases, autoimmune diseases, nephrotic syndrome, sarcoidosis, polychythemia vera, heroin addiction, Waldenstrom’s macroglobulinemia, cryoglobulinemia
Above 10 mg/ml - IgM monoclonal gammopathy, IgM myeloma; again, have to think cancer if elevated
What are the 2 forms of IgA?
IgA1: monomer; present in circulation
IgA2: dimer; predominant antibody in mucosal secretions and mucosal immunity
Structure of secretory IgA
Dimer
J chain
Secretory component (piece of peptide that comes from a protein called a poly-Ig receptor)
Formation of secretory IgA
Plasma cells secrete a dimeric IgA, which then binds to the Poly-Ig receptor on epithelial cells
Poly-Ig recpetor w/ the immunoglobulin attached is endocytosed into a vesicle which fuses w/ the membrane
Contents are released into the mucosal surface
During that process, Poly-Ig receptor is protealytically cleaved; a piece of the IgA receptor that was bound to the IgA2 goes w/ it
So, have IgA2 w/ this piece of Poly-Ig receptor that is called the secretory component
What is the purpose of the secretory component on secretory IgA?
Mucosal surface are where you contact the outsidenvironment; there are a lot of bacteria out there which secrete proteases
That secretory component keeps bacterial proteases from cutting it up before it can protect you
What type of IgA is serum IgA?
IgA1
What are normal serum concentrations of IgA?
2.1 +/- 0.5 mg/ml
What are abnormal IgA serum concentrations? What can they indicate?
Below 0.4 mg/ml - Immunodeficiency, monoclonal gammopathy of IgG or IgM
Above 4.5 mg/ml - IgA monoclonal gammopathy, infections, liver disease, autoimmune disease, cystic fibrosis, celiac disease, IgA nephropathy (verious serious, causes renal failure; IgA forms immune complexes and becomes caught in glomeruli of kidney, causing destruction) or Berger’s disease, sarcoidosis
Above 10 mg/ml - IgA monoclonal gammopathy, IgA myeloma; once again, have to think cancer
Characteristics of IgD
Relatively rare Usually found on cell surface Used as a marker of B cell maturation May play role as co-stimulator during antigen presentation May play tole in B cell development
What are normal serum concentrations of IgD?
Never seen in serum
See it on cells
Characteristics of IgE
Present only in trace amounts as a free monomer in the circulation
Binds to very high affinity Fc receptors on tissue mast cells and circulating basophils
Binds to moderate affinity Fc receptors on lymphocytes and monocytes
Primary antibody mediator of hypersensitivity, allergies, asthma
Primary anti-parasite antibody, sensitizes worms and other parasites for destruction by eosinophils
How are allergic reactions mediated by IgE?
Mast cells and basophils are full of granules that are full of vasoactive amines (histamines, serotonin)
IgE that is bound to Fc receptors will bring the 2 Fc receptors together and send signal inside cell to release the granules
Histamine and other substances that mediate allergic responses are then spilled out into the circulation
What are normal serum concentrations of IgE?
0.0003 mg/ml
What are abnormal IgE serum concentrations? What can they indicate?
Decreased IgE diagnostic for immunodeficiency, hypo- or agammaglobulinemia
Increased IgE diagnostic for allergic disorders, hypersensitivity disorders, parasitic infections, immunologic disorders, infectious mononucleosis, IgE myeloma
What region of the antibody is antigen binding? Component and cell receptor binding?
The 2 Fab fragments bind antigen
Fc fragment binds to Fc receptors and to complement
Antibody determinants
Isotypic determinants - determined by gene; shared by all members of a species (can raise an antibody that would interact w/ every IgG1 from any human)
Allotypic determinants - determined by allele; if there is an antibody that can bind to one epitope but not the other, can differentiate b/w strains A and B
Idiotypic determinants - determined by variable region; every antibody has a different idiotype
Monoclonal antibodies
Created by fusing spleen cells from an immunized mouse (w/ the desired antigen) w/ a mutant mouse myeloma cell line
Fused cells (hybridomas) are selected by culture
Immunogenicity
Ability to evoke an immune response
Antigenicity
Ability to bind an antibody
What is the difference b/w immunogenicity and antigenicity?
Immunogenicity is more than antigenicity
Many small molecules can bind tightly to antibodies but cannot activate B cell
How can antigenic small molecules be made immunogenic?
By attaching them to a large molecule (hapter-carrier)
Describe process for making an antigenic small molecule immunogenic
Create a carrier-hapten conjugate; carrier is a big protein
Inject it into an animal
Get antibodies to the carrier, antibodies to the hapten, and antibodies to the hapten-carrier conjugate
What are the properties of antigens that lead to immune response?
Foreigness (non-self structure) Molecular size (larger = more immunogenic) Chemical complexity (holopolymers usually poor immunogens while co-polymers of 2-3 different units may be immunogenic) Degradability (molecules that cannot be degraded and processed by APCs are poor immunogens; e.g. peptides containing D-amino acids)
What are other factors in immunogenicity?
Genetic constitution of immunized animal
Method of antigen administration
Amount of antigen administered
Use of an adjuvent to promote response
What are size considerations of antigens?
Under 1,000 Daltons: nonimmunogenic
1,000 - 6,000 Daltons : may or may not be immunogenic
Over 6,000 Daltons: usually immunogenic
Are carbohydrates antigenic?
Non-self carbohydrates (bacterial capsules, ABO blood group antigens) are often antigenic
Are lipids immunogenic?
Usually not immunogenic
Are nucleic acids immunogenic?
Poor immunogens themselves but good as haptens
Are proteins immunogenic?
Excellent antigens
Are metal ions immunogenic?
Only immunogenic as haptens
Are small organic compounds immunogenic?
Usually only immunogenic as haptens
Where is there greatest positional variability in amino acid composition of antibodies?
CDR1, CDR2, and CDR3 of variable region of heavy and light chain
What forms surface on antibodies where antigens interact?
CDR1, CDR2, CDR3
What type of interactions are antibody-antigen interactions mediated by?
Multiple noncovalent interactions, such as hydrogen bonds, ionic bonds, hydrophobic interactions, van der Waals interactions
Affinity
Describes the interaction of 2 molecular surfaces - one surface on the antigen (epitope) and one surface on the antibody (paratope)
Keq =
[AB]
_______
[Af][Bf]
Also called intrinsic affinity
Avidity
Interaction b/w an entire antibody (which can bind at least 2 epitopes) w/ an entire antigen (which can have thousands of epitopes)
Avidity is also called functional affinity
How does valency of interaction influence avidity of interaction?
Monovalent antibodies - low avidity of interaction
Bivalent antibodies - high avidity of interaction
Polyvalent antibodies - very high avidity of interaction
What properties of epitopes do antibodies recognize?
Composition
Conformation
Antibody specificity
A single antibody binds tightly to only a small number of 3 dimensional surfaces, which exist in a small number of antigens
Any change in the composition or conformation of those surfaces significantly reduces binding affinity
Shared epitopes
Near-identical 3 dimensional surfaces
Antibody cross-reactivity
An antibody that binds to one of the epitopes of one protein will often also bind to the shared epitope on another protein
How is cross-reactivity related to disease?
Some pathogen proteins contain an epitope that is very similar to an epitope present on a host protein
Some of the antibodies raised against the pathogens could cross-react and bind to the host protein
This can lead to autoimmune diseases
Cross-reactivity and myocarditis
Chamydia outer membrane protein/cardiac myosin heavy chain → myocaditis
Cross-reactivity and encephalomyelitis/multiple sclerosis
Hepatitis B viral protein/myelin basic protein → encephalomyelitis/multiple sclerosis
Cross-reactivity and myasthenia gravis
Herpesvirus protein/nicotinic acetylcholine receptor → myasthenia gravis
