Antibodies I & II - Diebel

Question 1

What is the H chain?

Answer 1

• 2 identical residue chains
• linked by disulfide bonds at the hinge region
• 5 kinds that define the class of antibody:
  
  • gamma
  • alpha
  • mu
  • epsilon
  • delta

Question 2

What is the L chain?

Answer 2

• 2 identical residue chains
• linked to heavy chain by disulfide bonds
• Two different types:
  
  • kappa
  • lambda

Question 3

What is a variable domain?

Answer 3

• domain that is different in sequence between antibodies of different specificities
• N-terminal of both heavy chain (V_H) and light chain (V_L)

Question 4

What is a constant domain?

Answer 4

• 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

Question 5

What are hypervariable regions?

Answer 5

• 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)

Question 6

What is the F_ab?

Answer 6

• 2 identical branches (split pieces of the “Y”) of an antibody cut at hinge region
• not linked
• Includes
  
  • 2 heavy chains
  • 2 light chains

Question 7

What is the F_C?

Answer 7

• bottom trunk/stalk of antibody if cut at the hinge region with papain
• Composed of:
  
  • 2 heavy chains

Question 8

What is the F_(ab’)2?

Answer 8

• two antigen-binding F(ab) portions linked together by disulfide bonds
  
  • retain some of hinge region

Question 9

What are the five classes of antibodies?

Answer 9

• 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
• IgD - three constant domains, only in B-cells
• IgE - four constant domains

Question 10

What is the function of IgG?

Answer 10

• neutralizes toxins and blood-borne viruses
• binds bacteria & facilitates their destruction by:
  
  • activating complement
  • binding them to phagocytic cells

Question 11

What is the function of IgA?

Answer 11

• present in mucosal areas
• secretory component protects it from proteolysis
  
  • allows for translocation of the dimer through epithelial layers

Question 12

What is the function of IgM?

Answer 12

• neutralizes toxins and blood-borne viruses
• binds bacteria & facilitates their destruction by:
  
  • very efficient complement activator
• first antibody to appear in serum after immunization

Question 13

What is the function of IgD?

Answer 13

• Uncertain
  
  • possiby functions mainly as a receptor on naïve B-cells

Question 14

What is the function of IgE?

Answer 14

• confers resistance to worms and other parasites
• causes Type I Immunopathology = immediate hypersensitivity/allergy

Question 15

What is antibody valence?

Answer 15

• number of antigenic determinants (epitopes) an antibody molecule can theoretically bine
  
  • IgG = 2
  • IgA = 4 (because it is a dimer)
  • IgM = 10 (because it is a pentamer)
  • F_ab = 1
  • F_(ab’)2 = 2
  • isolated VL = 0 (does not fxn alone)
  • isolated VH = 0 (does not fxn alone)

Question 16

What are antibody isotypes?

Answer 16

• subclasses of antibodies
  
  • e.g. IgG1, IgG2, IgG3, IgG4
• constant domain regions are slightly different, but the variable domain stays the same

Question 17

How many antibody isotypes are there all together in all five classes of antibodies?

Answer 17

10

Question 18

What are antibody allotypes?

Answer 18

• 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

Question 19

What are antibody idiotypes?

Answer 19

• unique combining region (variable regions)
• made up of the CDR amino acids of its Light and Heavy chains

Question 20

What is the relative concentration of IgG in the serum?

Answer 20

1000 mg/dL

Question 21

What is the relative concentration of IgA in the serum?

Answer 21

200 mg/dL

Question 22

What is the relative concentration of IgM in the serum?

Answer 22

100 mg/dL

Question 23

What is the relative concentration of IgD in the serum?

Answer 23

5 mg/dL

Question 24

What is the relative concentration of IgE in the serum?

Answer 24

0.02 mg/dL

Question 25

What part of antibodies does kappa and lambda refer to?

Answer 25

kappa refers to one of the constant light chain possibilities lambda refers to the other constant light chain possibility

Question 26

Which antibody isotypes have the highest serum half life?

Answer 26

* IgG = 8-23 days * IgA = 6 days * IgM = 5 days

Question 27

Which antibody isotype can cross the placenta?

Answer 27

IgG

Question 28

Which antibody isotype is the largest (molecular weight)?

Answer 28

IgM = 900 kD | (IgA = 150-600kD, IgE = 190kD)

Question 29

Which antibody isotype is the best for complement fixation?

Answer 29

IgM

Question 30

Which antibody isotype is the best for mast cell/basophil degranulation?

Answer 30

IgE

Question 31

Which antibody isotype is the best for bacterial lysis?

Answer 31

IgM

Question 32

Which antibody isotype is the best for antiviral activity?

Answer 32

IgA

Question 33

Which two antibody isotypes are best at toxin neutralization?

Answer 33

IgG & IgA

Question 34

What do elevated levels of IgM indicate?

Answer 34

* recent infection * other exposure to antigen

Question 35

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?

Answer 35

IgG

Question 36

Which antibody has a greater daily production than any other Ig isotype?

Answer 36

IgA

Question 37

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?

Answer 37

IgA

Question 38

What gene segments code for the variable domain region of heavy chain genes?

Answer 38

* V * J * D

Question 39

What gene segments code for the variable domain region of light chain genes?

Answer 39

* V * J

Question 40

Where does the constant region of the light chain come from in light chain synthesis?

Answer 40

* lambda light chain - only one C domain * chromosome 22 * kappa light chain - only one C domain * chromosome 2

Question 41

How is a heavy chain synthesized? | (Hint: 5 steps)

Answer 41

* bring one random D segment close to one J * DNA is cut * intervening DNA is discarged * remaining ends joined * bring a V segment up to the recombined DJ * DNA is cut * intervening DNA is discarged * 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

Question 42

What are the two enzymes that do the recombination of antibody and T-cell receptor DNA?

Answer 42

RAG-1 and RAG-2 recombinases

Question 43

How do RAG recombinases recombinate heavy chain DNA?

Answer 43

* 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 * splice

Question 44

Why do B-cells have randomizing mechanisms like Somatic Variation?

Answer 44

production of the V-D and D-J joints are "sloppy"

Question 45

What happens in Somatic Variation?

Answer 45

1. 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) 2. Cell can add a few nucleotides with an enzyme terminal deoxynucleotidyl transferase (TdT), which doesn't use a template so its additions are random

Question 46

What is the downfall of Somatic Variation?

Answer 46

* create a frame-shift mutation * abortively rearrange chains * incomplete antibody causes cell to die

Question 47

How does variation through Somatic Hypermutation work?

Answer 47

* 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

Question 48

When does class switching occur?

Answer 48

* 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.

Question 49

How does class switching occur?

Answer 49

* 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 V_H domain stays the same, but the C region of the heavy chain changes.

Question 50

What is allotypic exclusion?

Answer 50

* 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

Question 51

What is the role of allotypic exclusion in the generation of a functional B cell receptor (BCR)?

Answer 51

* 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

Question 52

How does RNA splicing and polyA site selection dictate what isotype of antibody is produced and whether that antibody is membrane bound or secreted?

Answer 52

* 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 #2 (membrane-bound gene) * instead use "S" Poly-A site #1 (secretory)

Question 53

What are the 12 stages of Ig expression and B-cell maturation?

Answer 53

* 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: IgM^high, IgD^low * alternative splicing to give both delta and mu chains * gains access to primary lymphoid follicle and matures * Mature naive B-cell: IgM^low, IgD^high * 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