Ab interaction w/ Ag Flashcards
What is an Ag?
A molecule on pathogen which triggers an Ab response
What do Ab recognise?
Recognise conformational Ags composed several sequentially discontinuous segments brought together by folding of molecule
What is different between Ab and TCR?
TCR can recognise linear Ag (sequentially linear segment of continuous residues) when presented by APC
What is an epitope?
Part of Ag recognised by Ab
Large Ag can have many different epitopes that are recognised by different Ab
What is a paratope?
Complementary part of Ab
What is the relationship between paratope and epitope?
They are confirmationally complementary - so they can bind together
Describe the structure of an Ab
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
What forms the Ag binding domain?
Variable region - heavy + light chain
How many segments of variability does each variable region have?
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
What are hyper-variable regions?
They corresponds to 3 loops @ outer edge beta sheets of Ig domain
Describe the Ag binding site
When CDR loops from heavy chain and light chain bought together, creates single hyper-variable site @ top each arm Ab molecule
What is combinatorial diversity?
Different combination heavy and light variable regions will generate different Ag specificities
What is significant about the framework regions?
Have less variability at any given amino acid position - more stable sequence
How is variability limited to the hyper-variable regions Ab?
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
What is the Ab-Ag interaction like?
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
What bonds are involved in the Ab-Ag interaction?
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)
What is the interaction between Ab and Ag determined by?
Dependent on Ab+Ag involved and distance between molecules
Total contribution of forces determine overall strength interaction
What is affinity?
Strength of interaction
Each force acts over short distance - 1x10^-7 mm or less
How can Ab affinity be calculated?
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
What is avidity?
Measurement of overall strength of Ab-Ag complex
Dependent on how many binding sites are occupied
What is valency?
Number of Ag binding sites available
All Ab are multivalent
Describe the different levels of valency
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
When is binding defined by affinity?
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
When is binding defined by avidity?
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
What is more important for functionality? (avidity or affinity)
Avidity likely to be more important than affinity
What else is used to strengthen an Ab-Ag interaction?
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
When does the first humoral response?
When B cell encounters an Ag, produces Ab to make up humoral response
What occurs during the primary immune response?
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
What occurs when the primary infection is cleared?
Small proportion B cells remain as memory B cells - these activated quickly when same pathogen encountered
What occurs in the secondary immune response?
Get a much higher level Ab production reached w/in shorter time frame
Maximal Ab secretion occurs 0-5days
IgG Ab most predominant
What occurs after the secondary infection is clear?
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
What are the functional consequences of Ab binding?
- Neutralisation microbes and toxins
- Opsonisation and phagocytosis microbes because Ab binding pathogen and subsequent binding to Fc receptor on immune cells
- Ab dependent cellular cytotoxicity
- Complement pathway - coating Ab-Ag complex w/ C1q
What can activation of the complement pathway cause?
- lysis microbes
- phagocytosis microbes opsonised with complement fragment eg. C3b
- Cleavage products - causes inflammation
Describe the neutralisation of viruses
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
How is vaccine efficacy measured?
Assessed by measuring circulating levels of neutralising Ab
Describe neutralisation of toxins
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
Describe opsonisation
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
Describe complement
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
What are Fc receptors?
How Ab interact with immune cells Several classes of Fc receptor based on Ab class they recognise
What is the g-chain responsible for?
Signalling molecule
Some Fc receptors associate with a g-chain
What occurs following Ab binding to the Fc receptor?
Aggregation occurs as aggregation receptors generate downstream signalling via ITAMa in associated chains
ITAMS - immunoreceptor Tyrosine based activation/inhibitory motifs
What are the effector functions induced by the Fc receptor?
Ab dependent cell cytotoxicity phagocytosis Apoptosis Mediator release Enhancement of Ag presentation
Describe the CD64 (FcYR1)
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
Describe the CD32 (FcYR2)
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
What does binding to FcYR2b cause?
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
Describe CD16 (FcYR3)
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
Describe the FCalphaR1 (CD89)
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
Describe the FcER1
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
Describe the CD23 (FcER2)
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
