Ab interaction w/ Ag

Question 1

What is an Ag?

Answer 1

A molecule on pathogen which triggers an Ab response

Question 2

What do Ab recognise?

Answer 2

Recognise conformational Ags composed several sequentially discontinuous segments brought together by folding of molecule

Question 3

What is different between Ab and TCR?

Answer 3

TCR can recognise linear Ag (sequentially linear segment of continuous residues) when presented by APC

Question 4

What is an epitope?

Answer 4

Part of Ag recognised by Ab

Large Ag can have many different epitopes that are recognised by different Ab

Question 5

What is a paratope?

Answer 5

Complementary part of Ab

Question 6

What is the relationship between paratope and epitope?

Answer 6

They are confirmationally complementary - so they can bind together

Question 7

Describe the structure of an Ab

Answer 7

Ab has heavy chain and light chain
Fc region responsible for effector function - comprised only heavy chain constant domains
Fab fragment - 1 constant + 1 variable region

Question 8

What forms the Ag binding domain?

Answer 8

Variable region - heavy + light chain

Question 9

How many segments of variability does each variable region have?

Answer 9

Each variable region has 3 segments of variability = hyper-variable region
Can identify hyper-variable region on heavy and light chain on H+L chain
Each Ab binding site have 6 hyper-variable regions = complementarity determining regions

Question 10

What are hyper-variable regions?

Answer 10

They corresponds to 3 loops @ outer edge beta sheets of Ig domain

Question 11

Describe the Ag binding site

Answer 11

When CDR loops from heavy chain and light chain bought together, creates single hyper-variable site @ top each arm Ab molecule

Question 12

What is combinatorial diversity?

Answer 12

Different combination heavy and light variable regions will generate different Ag specificities

Question 13

What is significant about the framework regions?

Answer 13

Have less variability at any given amino acid position - more stable sequence

Question 14

How is variability limited to the hyper-variable regions Ab?

Answer 14

Have much higher ratio of different amino acid at any given position relative to the most common amino acid
hence, variability limited to hyper-variable region

Question 15

What is the Ab-Ag interaction like?

Answer 15

Neither Ab or Ag is changed by binding
During interaction between Ab+Ag - 1 Ab binding site binds to 1 epitope on Ag

Binding is non-covalent and is reversible

Question 16

What bonds are involved in the Ab-Ag interaction?

Answer 16

Ionic bonds - occurs between oppositely charged amino acid side chains

H-bonds - H shared between electronegative O2/N atom

Hydrophobic bonds - groups interact together to exclude water w/ tyr, phe, leu, ile etc

Van der waals forces - occur when fluctuations in electron clouds surround molecules are oppositely polarised neighbouring atoms between transient poles (short range)

Question 17

What is the interaction between Ab and Ag determined by?

Answer 17

Dependent on Ab+Ag involved and distance between molecules

Total contribution of forces determine overall strength interaction

Question 18

What is affinity?

Answer 18

Strength of interaction

Each force acts over short distance - 1x10^-7 mm or less

Question 19

How can Ab affinity be calculated?

Answer 19

Can calculate because Ab-Ag reaction is reversible so can get dynamic eqm

K = [AbAg] conc. Ab/Ag complex
———- = ——————————–
[Ab][Ag] conc. unoccupied binding site on Ab
+ conc. unoccupied binding site Ag

K = affinity = eqm. constant
Can calculate how much AbAg complex present @ eqm

Typically higher affinity Ab will bind greater amount Ag in shorter period of time than lower affinity Ab

Question 20

What is avidity?

Answer 20

Measurement of overall strength of Ab-Ag complex

Dependent on how many binding sites are occupied

Question 21

What is valency?

Answer 21

Number of Ag binding sites available

All Ab are multivalent

Question 22

Describe the different levels of valency

Answer 22

Monovalent - can occur when low level Ag and not enough Ag present to occupy both Ag binding sites on Ab
Low avidity

Bivalent - Occurs when sufficient Ag so both Ag binding sites are occupied eg. IgG
High avidity

Polyvalent - IgM as can form pentameric structure of 5 IgM molecules - 10 possible Ab binding sites
Very high avidity

Question 23

When is binding defined by affinity?

Answer 23

Intrinsic affinity

Fab fragment:
Only 1 Ag binding site occupied
Valence = 1
Low eqm constant - 10^4 L/mol

IgG w/ insufficient Ag
Valency = 1, low eqm constant

Question 24

When is binding defined by avidity?

Answer 24

Functional affinity

IgG with sufficient Ag for both binding site to be occupied
Valency = 2
Higher eqm constant - 10^7 L/mol

IgM - up to 10

Question 25

What is more important for functionality? (avidity or affinity)

Answer 25

Avidity likely to be more important than affinity

Question 26

What else is used to strengthen an Ab-Ag interaction?

Answer 26

Additional signals such as Ig alpha and beta, once BCR ligated (accessory molecules) T cell help in form of T cells Additional co-stimulation required from co-stimulatory receptors eg. CD-19 All signals come together to induce proliferation 1000-10,000 times differentiation into plasma cells + Ab prod

Question 27

When does the first humoral response?

Answer 27

When B cell encounters an Ag, produces Ab to make up humoral response

Question 28

What occurs during the primary immune response?

Answer 28

1st infection Where naive B cells activated Begin to proliferate and differentiate into Ab producing plasma cells Maximal Ab production typically reached w/in 5-10 days Predominant Ab production is IgM Ab binds to Ag to eliminate pathogens encountered

Question 29

What occurs when the primary infection is cleared?

Answer 29

Small proportion B cells remain as memory B cells - these activated quickly when same pathogen encountered

Question 30

What occurs in the secondary immune response?

Answer 30

Get a much higher level Ab production reached w/in shorter time frame Maximal Ab secretion occurs 0-5days IgG Ab most predominant

Question 31

What occurs after the secondary infection is clear?

Answer 31

Following clearance, memory B cells generated at much higher number than following primary response Hence, why secondary vaccination important because increase pool memory cells able to respond to infection

Question 32

What are the functional consequences of Ab binding?

Answer 32

1. Neutralisation microbes and toxins 2. Opsonisation and phagocytosis microbes because Ab binding pathogen and subsequent binding to Fc receptor on immune cells 3. Ab dependent cellular cytotoxicity 4. Complement pathway - coating Ab-Ag complex w/ C1q

Question 33

What can activation of the complement pathway cause?

Answer 33

1. lysis microbes 2. phagocytosis microbes opsonised with complement fragment eg. C3b 3. Cleavage products - causes inflammation

Question 34

Describe the neutralisation of viruses

Answer 34

Depends on type of virus, target cell and class Ab Limit viral infectivity - may inhibit virus-cell interaction Prevent endocytosis virus Prevent uncoating inside virus All above methods more effective with complement

Question 35

How is vaccine efficacy measured?

Answer 35

Assessed by measuring circulating levels of neutralising Ab

Question 36

Describe neutralisation of toxins

Answer 36

Ab can bind bacterial endotoxins Prevents binding to surface receptors immune cells eg. binding cholera toxin to ganglioside GM1 Stimulates toxin clearance in Fc receptor mediated manner IgG+IgA - important neutralising Ab

Question 37

Describe opsonisation

Answer 37

Coat particles by either Ab, complement, acute phase proteins (APP) - CRP Ab bind micro-organism via Fab fragment and binds to cell via Fc Does on phagocytes eg. macrophages to cause phagocytosis Increases efficiency of phagocytic process - organism cleared more effectively

Question 38

Describe complement

Answer 38

Ag-Ab complex can activate complement system Part of innate immune response - classical and alternative pathway ``` Functional pathways: Chemotaxis Opsonisation Lysis of target cells Priming adaptive immune response ```

Question 39

What are Fc receptors?

Answer 39

``` How Ab interact with immune cells Several classes of Fc receptor based on Ab class they recognise ```

Question 40

What is the g-chain responsible for?

Answer 40

Signalling molecule | Some Fc receptors associate with a g-chain

Question 41

What occurs following Ab binding to the Fc receptor?

Answer 41

Aggregation occurs as aggregation receptors generate downstream signalling via ITAMa in associated chains ITAMS - immunoreceptor Tyrosine based activation/inhibitory motifs

Question 42

What are the effector functions induced by the Fc receptor?

Answer 42

``` Ab dependent cell cytotoxicity phagocytosis Apoptosis Mediator release Enhancement of Ag presentation ```

Question 43

Describe the CD64 (FcYR1)

Answer 43

Binds monomeric IgG1 + IgG3 w/ high affinity IgG4 w/ low affinity No binding to IgG2 3 extracellular domains allows binding to sole IgG monomer intracellular portion associates with Y chain for ITAM signalling Expressed on mononuclear phagocytes

Question 44

Describe the CD32 (FcYR2)

Answer 44

Comes in 2 forms: FcR2a - wide cellular distribution moderate affinity for monomeric IgG1 + IgG3 high affinity for complexed IgGs Has ITAM intracellular portion - binding to receptor is activatory FcR2b - same specificity for IgG BUT instead has ITIM = binding is inhibitory

Question 45

What does binding to FcYR2b cause?

Answer 45

Ab conc reaches threshold level, Ab-Ag complex binds to inhibitory receptors eg. Fc2b simultaneously w/ BCR Causes negative feedback which blocks BCR signalling and ultimately inhibiting B cell responses

Question 46

Describe CD16 (FcYR3)

Answer 46

2 forms - A = transmembrane molecule and B = GPI linked molecule FCY3a - moderate affinity for monomeric IgG Associated w/ Y, Beta and zeta chains - CD3 complex expressed monocytes, macrophages, NKC, some T cells Responsible for ADCC FCY3b - GPI linked w/ low affinity for IgG Expressed neutrophils and basophils activated by lipid raft formation and associates with other membrane signalling molecules

Question 47

Describe the FCalphaR1 (CD89)

Answer 47

``` Associated gamma signalling chain Found on myeloid cells Composed 2 extracellular Ig-like domains Can bind to both IgA + IgA2 Ab Can trigger phagocytosis, cell lysis + release inflammatory mediators ```

Question 48

Describe the FcER1

Answer 48

High affinity for IgE 2 Ig-like domains - associate w/ gamma and beta chains Expresses mast cells and basophils X-linking Ab molecules bound to R1 -> mediator release histamines IgE always bound FcER1 .˙. always saturate (low serum IgE) - allergic response is quick

Question 49

Describe the CD23 (FcER2)

Answer 49

Low affinity receptor for IgE Not member Ig-superfamily similar to c-type lectins eg. mannose binding lectin Expressed on leukocytes and lymphocytes in 2 forms: CD23a - B cells, involved IgE prod CD23b - expressed on lots of cell types and induced by IL-4