Antibodies - Diebel Flashcards
What is the H chain?
2 identical residue chains
linked by disulfide bonds at the hinge region
5 kinds that define the class of antibody:
gamma
alpha
mu
epsilon
deltaWhat is the L chain?
2 identical residue chains
linked to heavy chain by disulfide bonds
Two different types:
kappa
lambdaWhat is a variable domain?
Antigen Binding Sites
domain that is different in sequence between antibodies of different specificities
N-terminal of both heavy chain (VH) and light chain (VL)
What is a constant domain?
region of each chain type that is essentially identical no matter what the specificities of the antibodies are
1 in light chains up to 4 in heavy chains
What are hypervariable regions?
most amino acid sequence variability in the V domain is in 3 areas = hypervariable regions
amino acids in the hypervariable regions comprise the actual antigen-binding site Complementarity-determining regions (CDRs)
What is the Fab?
2 identical branches (split pieces of the "Y") of an antibody cut at hinge region
not linked
Includes
2 heavy chains
2 light chainsWhat is the FC?
bottom trunk/stalk of antibody if cut at the hinge region with papain
Composed of:
2 heavy chains
What is the F(ab’)2?
two antigen-binding F(ab) portions linked together by disulfide bonds
retain some of hinge region
What are the five classes of antibodies?
IgG - three constant domains in heavy chain
IgA - three constant domains, forms dimer connected by J-chain, with secretory portion
IgM - four constant domains, forms pentamer, connected by J-chain
IgD - three constant domains, only in B-cells, membrane bound
IgE - four constant domains, only secreted in monomeric form
What is the function of IgG?
neutralizes toxins and blood-borne viruses
binds bacteria and facilitates their destruction by:
activating complement
binding them to phagocytic cells
What is the function of IgA?
present in mucosal areas
secretory component protects it from proteolysis
allows for translocation of the dimer through epithelial layers
What is the function of IgM?
neutralizes toxins and blood-borne viruses
binds bacteria and facilitates their destruction by:
very efficient complement activator
first antibody to appear in serum after immunization
has not gone through class switching recombination
What is the function of IgD?
Uncertain
possiby functions mainly as a receptor on naïve B-cells
What is the function of IgE?
confers resistance to worms and other parasites
causes Type I Immunopathology = immediate hypersensitivity/allergy
What is antibody valence?
number of antigenic determinants (epitopes) an antibody molecule can theoretically bind
IgG = 2
IgA = 4 (because it is a dimer)
IgM = 10 (because it is a pentamer)
Fab = 1
F(ab')2 = 2
isolated VL = 0 (does not fxn alone)
isolated VH = 0 (does not fxn alone)
What are antibody isotypes?
subclasses of antibodies
e.g. IgG1, IgG2, IgG3, IgG4
constant domain regions are slightly different, but the variable domain stays the same
How many antibody isotypes are there all together in all five classes of antibodies?
10
What are antibody allotypes?
minor allelic differences in the sequence of immunoglobulins between individuals
determined by the allotypes of your parents (Mendelian fashion)
e.g. comparing two IgG1 molecules, but one is from a gene from one parent, and the other is from a gene from the other parent
What are antibody idiotypes?
unique combining region (variable regions)
made up of the CDR amino acids of its Light and Heavy chains
What is the relative concentration of IgG in the serum?
1000 mg/dL
What is the relative concentration of IgA in the serum?
200 mg/dL
What is the relative concentration of IgM in the serum?
100 mg/dL
What is the relative concentration of IgD in the serum?
5 mg/dL
What is the relative concentration of IgE in the serum?
0.02 mg/dL
What part of antibodies does kappa and lambda refer to?
kappa refers to one of the constant light chain possibilities
lambda refers to the other constant light chain possibility
Which antibody isotypes have the highest serum half life?
IgG = 8-23 days
IgA = 6 days
IgM = 5 days
Which antibody isotype can cross the placenta?
IgG
Which antibody isotype is the largest (molecular weight)?
IgM = 900 kD
IgA = 150-600kD, IgE = 190kD
Which antibody isotype is the best for complement fixation?
IgM
Which antibody isotype is the best for mast cell/basophil degranulation?
IgE
Which antibody isotype is the best for bacterial lysis?
IgM
Which antibody isotype is the best for antiviral activity?
IgA
Which two antibody isotypes are best at toxin neutralization?
IgG & IgA
What do elevated levels of IgM indicate?
recent infection
other exposure to antigen
Which antibody can be used as a blocking antibody to block TNF production (as in rheumatoid arthritis) or to block allergens (can out-compete IgE to desentize a hypersensitivity reaction?
IgG
Which antibody has a greater daily production than any other Ig isotype?
IgA
Which antibody is present at very high levels in colostrum, is present in breast milk, and provides an excellent level of protection to newborns against respiratory and intestinal infections?
IgA
What gene segments code for the variable domain region of heavy chain genes?
V
J
D
What gene segments code for the variable domain region of light chain genes?
V
J
Where does the constant region of the light chain come from in light chain synthesis?
lambda light chain - only one C domain
chromosome 22
kappa light chain - only one C domain
chromosome 2
How is a heavy chain synthesized?
Hint: 5 steps
bring one random D segment close to one J
DNA is cut
intervening DNA is discharged
remaining ends joined
bring a V segment up to the recombined DJ
DNA is cut
intervening DNA is discharged
remaining ends joined
entire VDJ region is assembled
VDJ region is joined with constant region of particular antibody through splicing
final mRNA product can be transcribed
What are the two enzymes that do the recombination of antibody and T-cell receptor DNA?
RAG-1 and RAG-2 recombinases
How do RAG recombinases recombinate heavy chain DNA?
recombinases first bind splice signals to the right of a D segment and the left of a J segment
pull them together (synapsis)
cut (cleavage)
splice (joining)
recombinases bind splice sequence to the right of a V segment and the left of a D segment
pull them together
cut
spliceWhy do B-cells have randomizing mechanisms like Somatic Variation?
production of the V-D and D-J joints are “sloppy”
What happens in Somatic Variation?
Exonucleases chew away a few nucleotides after the DNA is cut but before two gene segments are joined (before D joined to J, or before V joined to DJ)
Cell can add a few nucleotides with an enzyme terminal deoxynucleotidyl transferase (TdT), which doesn’t use a template so its additions are random
What is the downfall of Somatic Variation?
create a frame-shift mutation
abortively rearrange chains
incomplete antibody causes cell to die
How does variation through Somatic Hypermutation work?
Activation-Induced Deaminase (AID) converts random cytosines→uracil in the CDR gene regions of one individual antibody
C:G pair becomes uracil:guanine mismatch
Uracil bases are excised by the repair enzyme uracil-DNA glycosylase
DNA polymerases then fill in the gap
create mostly single-base substitution mutation
One daughter makes different antibody at the end of cell division
might be better OR worse
When does class switching occur?
After activation:
B-cells switch from membrane-bound IgM and IgD to → secreted IgM by differential splicing
As B-cells continue to divide:
class switch to production of IgG by DNA rearrangement
Any time:
activated B-cells may continue to class switch to produce IgE or IgA by further DNA rearrangement.
How does class switching occur?
Cell with particular H-chain VDJ combination + mu (IgM) and delta (IgD) genes goes back to its DNA form
loops-out mu and delta
puts the H-chain VDJ combination next to different C region gene (gamma (IgG), epsilon (IgE), or alpha (IgA))
excises/discards intervening DNA
cannot go back and express the constant regions of IgM or IgD anymore
The L-chain and the VH domain stays the same, but the C region of the heavy chain changes.
What is allotypic exclusion?
Only one heavy chain (maternal/paternal) and one light chain (either kappa/lambda, either maternal/paternal) are synthesized in any one B-cell.
All the other genes are silenced.
Though the person can make two allotypes, each individual B-cell makes only one.
In somatic variation, sloppy recombination often ends up with a frame-shift mutation
cell can “try again” with the other allele
even though any single B-cell is theoretically capably of making 2 heavy chains (maternal/paternal) and 4 light chains (maternal/paternal & kappa/lambda), that never happens
it makes only one of each and all other alleles are excluded
What is the role of allotypic exclusion in the generation of a functional B cell receptor (BCR)?
allows progenitor B-cell to make two attempts at producing a productive allele
progenitor B-cell x2 attempts (ma/pa)
pre-BCR (mu) x2 attempts (ma/pa)
IgM (mu + kappa) x2 attempts (ma/pa)
mu + lambda x2 attempts (ma/pa)
if cell still not successful (all four combinations failed) → leads to cell death
How does RNA splicing and polyA site selection dictate what isotype of antibody is produced and whether that antibody is membrane bound or secreted?
As mature B-cells are activated to divide and differentiate by their cognate antigen
they switch from membrane-bound IgD and IgM to → secretory IgM
switch occurs at the level of processing mRNA transcripts
splice out “M1/M2” Poly-A site (membrane-bound gene)
instead use “S” Poly-A site (secretory)
What are the 12 stages of Ig expression and B-cell maturation?
Stem Cell (μ germline, κ/λ germline)
Early pro-B cell (μ germline, κ/λ germline)
Late pro-B cell (μDJ, κ/λ germline)
Large pre-B cell (μVDJ, κ/λ germline)
Small pre-B cell (μVDJ, κ/λ germline)
Immature B-cell (μVDJ, κ/λ VJ)
Imature B-cell: IgM+, IgD-
leaves bone marrow and enters peripheral circulation
Immature B-cell: IgMhigh, IgDlow
alternative splicing to give both delta and mu chains
gains access to primary lymphoid follicle and matures
Mature naive B-cell: IgMlow, IgDhigh
enters circulation and binds specific antigen in lymphoid tissue draining infection
Antigen-activated B-lymphoblast
Alternative splicing to secrete Ig
Isotype switching
Somatic hypermutation
Antibody-secreting plasma cell
fighting the current infection
Memory cell
preparing for future infection
What does low albumin signify?
Malnutrition or Liver Disease
What is the “M” peak associated with?
Multiple Myeloma - differentiated B-cells are multiplying like crazy through monoclonal expansion.
If you were missing the gamma peak on an antibody electrophoresis, then you would know that you are missing what?
B-cells
What are the 3 gene segments for the heavy and light chains?
Heavy Chain: Chromosome 14
Lambda Light Chain: Chromosome 22
Kappa Light Chain: Chromosome 2
Immunoglobulin Domains
Two or more B-pleated sheets arranged in opposite directions that are stabilized by one or more disulfide bonds.
Which antibody isotypes are the smallest (molecular weight)?
IgD and IgG = 150 kD
Which antibody cross-links on the surface of mast cells or basophils causing the release of histamine and the synthesis of prostaglandins and leukotrienes?
IgE
Which antibody plays a major role Parasitic and Fungal infections?
IgE
Clonal Selection Theory
Each cell of the immune system is programmed to make ONLY ONE antibody and the choice is random. The best-fitting clones are selected by antigen.
Define Recombination
Changing the relative positions of two pieces of DNA (bringing one of many V’s together with the correct C to copy DNA into mRNA)
–>Splicing at the DNA level
Omenn Syndrome
RAG recombinases get knocked out and neither B nor T cells are made
Receptor Editing
When faulty rearrangement is detected (like a stop codon is generated) or when an anti-self receptor is displayed, the RAG recombinases can “try again” if they are still active with the same gene.
As B-cells continue to differentiate, they can undergo additional class switching from IgM to IgG, IgA, or IgE which occurs at the level of…
Rearrangements of DNA using RAG1 and RAG2
Recombined V(D)J units are hypermutable, which means…
Each time a B-cell divides after antigenic stimulation, there is a good chance that one of the daughters will make a slightly different antibody.
Define Affinity Maturation
Selection and best-fitting mutations after antigenic stimulation allows a gradual increase of affinity during an immune response
B-Cell proliferation cytokines
IL-2
IL-4
IL-5
secreted from activate Th-Cells
B-Cell Differentiation (Class Switching) Cytokines
IL-2 IL-4 -->IgG1, IgE IL-5 -->IgE IFN-gamma --> IgG2a TGF-Beta
Antagonism Cytokine for B-Cells
IFN-gamma (blocks class switching to IgE by IL-4)
RAG1 and RAG2 function as lymphoid-specific recombinases and are responsible for …
VDJ Recombination and Class-Switching Recombination (no overlap in timing)
TdT is responsible for …
Somatic Mutations via N-nucleotide additions
