Antibodies - Li 4/5/16 Flashcards Preview

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Flashcards in Antibodies - Li 4/5/16 Deck (23):
1

basic definitions

  • antigen
  • antibody

relationship between teh two

antigen : any substance (exog from environment or endog from within body) that generates an immune response → generation of antibodies

antibody aka immunoglobulin : protein produced by B cells that tags microbes or infected cell to either mark for immune system attack or directly neutralize

  • paratope (region that binds antigen) binds epitope region on antigen

2

antibody structure

four polypeptide chains : 2 identical heavy, 2 identical light

  • disulfide bonds connect heavy-heavy and each heavy-light

heavy and light chains have 4 and 2 domains respectively

  • N terminal domains (VL and VH) are variable domains
  • other domains (CL on light; CH1, CH2, CH3 on heavy) are constant domains
  • heavy chain hinge domain (H) allows two arms of Ig to move relative to each other

3

diversity of Ig molecules

due to variability of V domains

 

variable domains are formed by antigen-binding (aka "combining") sites

  • comprised of framework regions and 3 CDRs (complementarity-determining regions)
    • CDRs: loops of polypep at ends of paired VH and VL domains, aka hypervariable regions
    • framework regions: mostly beta sheet, hold CDRs in place
  • CDRs of VH and VL make up the combining site; held together by interactions between CH1 and CL domains

4

paratope-epitope binding

 

what happens with large antigens?

  • each Ig has 2 identical paratopes (comprising one heavy + one light chain) 
  • epitopes (aka antigenic determinant) typically have 15-25 a.a. side chains that form approx 50-200 interactions with the antibody paratope
  • paratopes and epitopes are sterically and chemically complementary via multple noncovalent bonds
    • small mol? pit. long mol? groove. larger surface? lumpy surface with chemical complementarity.

large complex antigens have many epitopes

  • each Ig is epitope-specific, but the population of Ig as a whole can bind the many epitopes on an antigen → process of antibody production in response to a complex antigen = polyclonal response
  • best binding epitopes may direct bulk of antibody production → known as immunodominant epitopes

5

protease cleavage of antibodies

  • what happens
  • fragments produced

proteases cleave antibodies at specific sites, produce...

Fab : univalent antigen-binding gragments (1 Ig light chain + amino terminal part of 1 Ig heavy chain, linked by disulfide bond)

  • contains variable part of Ig, which contains paratope and first constant region

Fc : crystalizable fragments made of carboxy-term parts of heavy chain constant regions of heavy chains, linked by disulfide bonds

6

papain

protease

 

antibody → 2 Fab fragments + 1 Fc fragment

7

pepsin

protease

antibody → 1 (Fab')2 fragment + fragments of heavy chain

  • (Fab')2 includes 2 light chains and parts of 2 heavy chains, incl the hinge region

8

Ig gene complexes

  • location
  • contents

gene complexes for Ig chains occur in segments

  • heavy chain genes in chr14q32.3
  • light chain genes in chr2p11.2, 22q11.2

 

each segment has multiple versions of constant (CH), joint (JH), diversity (D), and variable (VH), some fxal and some pseudogenes

 

*Ig light chain → either kappa or lambda chain

9

somatic recombination of Ig genes

humans can make 1000B variants of antibodies, but only have 30k genes...how?

V(D)J recombination

during devpt of B cells, H and L chain genes are assembled from libraries of shorter gene segments (germ-line configuration → cutting out lots of DNA to form B cells that produce antibodies of a specific idiotype)

 

TCRs resemble antibody Fab regions → TCR genes are assembled by same mechanism!

  • alpha chain : V and J segments
  • beta chain V, D, J segments

10

V(D)J recombo specifics

  • where/how splicing takes place
  • proteins that make it happen

conserved sequences define DNA cut/splice sites

  • conserved heptamer and nonamer, with spacing of specific length
  • V-23bp...12bp-D-12bp...23bp-J
  • splicing machiner requires 12bp and 23bp spacers in order to make cut → precludes splicing of V directly to J!

V/D and D/J splice joints occur in CDR3 region → brings diversity straight to the combining sites!

 

RAG1 and RAG2 (Recombo of Antibody Genes) are found only in lympocytes, possible bacterial ancestry

  • RAG1/RAG2 gene pdts recognize conserved seqs and target initial cuts

proteins resp for splicing are in all cells, part of DNA repair machinery

  • some DNA-repair defs can lead to immunodef!

11

creating additional diversity in Ig gene formation

(D)

V/D and D/J splice joints occur in CDR3 region → brings diversity straight to the combining sites!

  • DNA terminal deoxyribonucleotidyl transferase (TdT, "terminal transferase") 

    randomly adds nucleotides to cut ends of V, D, J segments

  • TdT only found in developing lymphocytes!

12

affinity maturation

repeated exposure to antigen results in progressive increase in antibody affinity

  • booster shots! improve affinity of antibody for antigen

 

basis: in germinal centers, there's lots of somatic mutation of variable regions of Ig genes

  • mutation is random...
    • some will lower affinity for antigen → cells will die
    • some will increase affinity for antigen → proliferate extensively, forming increasing fraction of pop
  • net effect: average antibody affinity rises (low affinity die, high affinity prolif)

13

consequences of errant antibody gene rearrangement

VDJ rearrangement gone wrong can join VDJ region to somewhere other than constant region (i.e. another gene or another chromosome)

  • this can place the joined gene under powervul B-cell specific transcriptional enhancer, which is near VDJ region...
  • if the gene is involved in control of cell prolif → uncontrolled cell division, formation of clone of malignant cells :(

ex. Burkitt lymphoma

14

5 classes of Ig

subclasses

heavy chain defines the class/isotype of antibody

  • IgA, IgG, IgM, IgE, IgD

each have distinct biological props

 

subclasses - determined by diffs between heavy chains

IgG has 4 subclasses

IgA has 2 subclasses

15

monoclonal antibodies

recognize a single epitope

  • each ab producing cells makes only one species of heavy chain and one species of light chain → monoclonal

therefore, a complex antigen generates a polyclonal response 

  • might end up with lower specificity with a mixture of antibodies, since the likelihood of at least one epitope being found on another molecule is higher

16

making antibodies in lab setting

  • ab-producing cells don't live long in culture → immortalized by fusing with tumor cell lines, which are then cloned and screened for production of desired ab
  • monoclonal antibodies are made in mice, but human immune system will recognize mouse-made ab as foreign
    • solution: DNA encoding CDRs of V domain can be cloned, sequenced → incorporated into human H and L genes → those genes are introduced into mouse cells, which make humanized antibodies (less immunogenic)
  • genetic engineering can make engineered antibodies that contain parts of ab chains → serve as targeting mechanisms for drug delivery

17

allelic exclusion

successful assembly of one heavy-chain gene or one light-chain gene on a chromosome prevents rearrangement on the other

 

ex. if a mouse is genetically engineered such that DNA contains pre-assembled heavy- and light-chain genes → rearrangement of endogenous genes is suppressed!

  • virtually all B cells produce same ab. not good for mouse...good for science

also works for T lymphocytes

18

B cells → plasma cells

  • diffs in Ig production

B cells make Ig as a transmembrane protein

  • key exons coding transmembrane and cytosolic domains are retained

plasma cells (once matured from B cells) make same Ig but secrete it!

  • key exons are excised

19

primary and secondary response

  • diffs in responses
  • diffs in antibodies

primary response: response to antigen that immune system has never encountered

  • dominated by IgM
  • low affinity antibodies

secondary response: response to an antigen previously encountered

  • dominated by IgG (via class switching)
  • high affinity antibodies
  • larger, longer, faster, more sensitive (lower dose req) than primary responses! due to proliferation of cells binding antigen following first exposure
    • single downside...cells produced are short-lived → why you should get booster shots!

20

serological diagnosis of infection

specific immune response can be used to diagnose infection

  • caveat: "immunological memory" exists → just because you see an antibody present, doesnt mean there's an active infection
  • however, if immune response is dominated by IgM → recent exposure/infection indicated

also applies to newborns: get all antibodies mom produces as IgG (which can cross placenta)

  • any IgM that's present had to have been made in utero → infection!

21

class switching

 

  • what is it
  • how does it happen
  • key features (reversibility, regulation)

effected by splicing of DNA in constant region (diff DNA markers and mechanisms than VDJ recombo)

 

first antibody made in an immune response is IgM...that can change with a class switch

  • VDJ segments encoding VH domain are retained
  • segments encoding IgM constant regions are deleted
  • VDJ exon becomes associated with DNA that encodes constant region of IgG or another class

net resultcombining site specificity stays the same, but isotype changes!

 

*new isotype is determined by size of DNA segment deleted since heavy chain constant regions are adjacent to one another, downstream from VDJ exon

*class switching is irreversible because DNA is deleted  → can't be regained

*process is regulated by cytokines, which activate transcripton of particular S regions (sites of switching)

22

how do you distinguish monoclonal and polyclonal lymphocyte pops?

Southern blotting

 

monoclonal pop → might rep a malignant or premalignant state

polyclonal pop → might rep an inflammatory process

23

7 ways to generate all the antibody diversity we see

1. diff combos of heavy/light chains

2. VDJ recombo

3. jx diversity (RSS - flanking segments can vary)

4. alt RNA splicing

5. class switch recombo

6. somatic hypermutation

7. genetic variation (maternal/paternal)