antibody structure

Question 1

how is immunity classified?

Answer 1

innate and adaptive

• innate is the first response, quicker but no memory
• adaptive can be split into humoral (antibodies) and cell-mediated (cytotoxic T cells, phagocytes)

Question 2

what makes antibodies?

Answer 2

B lymphocytes

Question 3

BCR is a…?

Answer 3

surface bound antibody

Question 4

how many diff antibody specificities does the human body have?

Answer 4

10^8

Question 5

antibody structure

Answer 5

consists of 4 polypeptide chains, 2 identical heavy chains and 2 identical light chains.

chains held together by disulphide (S-S) bridges - stabilised

the antibodies are glycosylated, which makes a particular type of confirmation

Fab (determines antigen binding specificity) and Fc region (biological activity, complement will bind within here)

hinge region for flexibility

Question 6

fab vs fab2?

Answer 6

F(ab’)2 and Fab, are antigen-binding fragments that can be generated from the variable region of IgG and IgM, by protease digestion

F(ab’)2 fragments consists of 2 antigen-binding regions joined by hinge through disulfides. This fragment is void of most of the Fc region.

Question 7

domains?

Answer 7

Fab2 and Fc region have individual domains - domains can be duplicated to produce variability

Question 8

Avidity

Answer 8

the overall strength of binding between an antibody and an antigen (sum of all the binding sites)

Question 9

Humoral immunity

Answer 9

immunity mediated by macromolecules found in extracellular fluids such as secreted antibodies, complement proteins, and certain antimicrobial peptides.

Question 10

Cell-mediated immunity

Answer 10

immune response that does not involve antibodies, but rather involves
the activation of phagocytes, antigen-specific cytotoxic T-lymphocytes, and the release of various
cytokines in response to an antigen.

Question 11

Fab2 region

Answer 11

constitutes the antigen binding region

• Antigen recognition site composed of the variable region of both the heavy and light chain
• This determines both the specificity and the affinity and avidity of the interaction with antigen.

Question 12

Fc region?

Answer 12

It’s the crystallizable fraction. Gives Ab its function.

• Fc region confirms the functional properties of antibody.
• Recognised by FcR (Fc receptors)
• Binds complement

Question 13

how do antibody classes differ?

Answer 13

differ depending on their heavy chain constant region
9 subclasses of antibody in humans
4 gamma
1: mu, epsilon, delta
2: alpha

Question 14

antibodies have 4 different functions - what are they?

Answer 14

1. immune complex, complement classical pathway (C1q)
   - antigen binds to fab2 regions forms complex which complement can bind to
2. opsonisation + phagocytosis
   - antibody binds to antigen, and antibody can bind to the Fc receptor on phagocytic cells
3. target cell
   - Fc receptor on killer cells - infected cell with antigen on surface
4. primed cell, sensitisation
   - IgE produced in first encounter, second encounter the IgE binds to antigen and mast cells degranulate, histamines and anaphylaxis

Question 15

IgM

Answer 15

• pentameric, 5 linked by J chain which stabilises the whole structure
• 4 heavy chain constant regions
• first class of Ab to evolve in the primary response, low affinity but high avidity overall –> ROUGH and READY, binds to lots of stuff but just weakly
• the IgM monomers mainly forms the BCR
• activates classical complement pathway

Question 16

IgG

Answer 16

monomeric, heavy chain with 3 constant regions

highest conc in serum (circulating) (IgG 1, 2, 3, 4 subclasses)

can cross placenta and protect foetus

activates classical complement pathway

opsonisation

IgG has a long half life, once primed to antigen, IgG is secondary antibody produced

IgG split half and half between vasculature and outside
venous system. 50% of IgG is in tissues.

Question 17

IgA

Answer 17

lots of diff polymers, in humans mostly monomers and dimer and 1 J chain per polymer

next highest circulating conc after IgG

2 subclasses, IgA1 in serum, IgA2 in mucous

found in milk, sweat, saliva, tears

lines mucosal defences

does not activate complement

Question 18

how does IgA become secretory IgA?

Answer 18

transported across mucosal epithelium from the basolateral surface to the luminal surface

IgA dimer binds to a poly-Ig receptor and forms a complex with it. this gets endocytosed into the cell and enclosed in a transport vesicle. the receptor gets cleaved by proteolysis and the IgA dimer with new secretory component is released - now secreted IgA!

Question 19

IgD?

Answer 19

like IgG but a few diff in heavy chain constant region

specific antigen binding sites but no effector function, expressed on B cell surface with monomeric IgM

very low serum conc

Question 20

IgE?

Answer 20

heavy chain - 4 constant regions

Majority bound to mast cells and basophils through high affinity FcεR1

involved in defence against parasitic infections, eg. helminths - mast cells good at killing multicellular organisms

Trace levels in serum (elevated in allergic or heavy parasitic
infection)

Question 21

surface bound Ig?

Answer 21

this Ig has a short intracytoplasmic tail, therefore needs accessory molecules to form the BCR - CD79 which forms a complex with the BCR after antigen binding, and is made of 2 chains: CD79a and CD79b

most B cells express monomeric IgM or IgD, both have same specificity on each cell

(some tissue bias, e.g. mucosal B cells express IgA)

Question 22

what does BCR ligation (recognition) lead to?

Answer 22

leads to ITAM phosphorylation (immunoreceptor tyrosine based activation motifs), which leads to downstream cascade resulting in differentiation into plasma cell and antibody production.

results in clonal expansion where the B cell activated gets cloned and more copies of it are made. small % live as memory cells, majority become effector cells (plasma cells) which produce antibody. plasma cells have a short lifespan and apoptose after a few days.

Question 23

class switching?

Answer 23

f1st Ab response is IgM, class switching occurs and other classes made, esp IgG.

somatic hypermutation (mutations of variable region gene sequences) which increases affinity of binding site for antigen and affinity maturation

Question 24

how are antibodies used in clinic?

Answer 24

monoclonal antibodies!
developed by Kohler and Milstein in 1975

Inject the antigen you want to make antibodies against into the animal, and harvest the B cells from spleen after a few weeks.

Fuse B cells with myeloma cells (constantly
growing lymphoid cells). Myeloma cells are immortal cells from a B cell tumour, they lack the HGPRT gene.

Ethylene glycol is used to help fuse - melts membranes. Forms hybidroma which are the only cell type that can grow in the HAT medium.

The hybridoma will spontaneously grow like myeloma. And also produce Ab.

Question 25

main function of monlocnal antibodies?

Answer 25

anti inflammatory or anti cancer

Infiximab used for athrities (anti TNF-alpha).

TNF-a = inflammatio, partcuilalry in joints. Helped
athritis a lot. Mops up inflam mediators, causing disease, (so doesn’t realy stop disease)

Rituximab binds to molecule found on B cell, to treat B cell tumour.

Herceptin – binds to receptor for growath factor.

Zumab – humanized molecule antibody has a bit of change, to look like human molecule.

Ximab at the end = chimeric antibody. (part of human and mouse antibody – they do this to stop it
from becoming an antigen itself)

new checkpoint inhibitor: Nivolumab is anti-PD1, stops switching immune system off.

Question 26

Problems with using antibodies as a therapy

Answer 26

Constant region heterogeneity. If have mouse Ab against TNF. Constatn region of that Ab will be of
murine nature (not human) so get antibody aginst it (anti antibody repsonse). That can cause inflammation

 Most early McAb were murine
 Anti-antibodies
 Inflammation

Solutions
 Chimeric antibodies: Murine antigen recognition sites grafted to human constant regions…. humanised.

 Fully humanised antibodies: using ‘phage display (in vitro McAb) and humanised mice!

 To deal with it, spliced murine with human anitbody. Take murine Fab2 region (target the
antigen) and joined with human constant region. (minimise the response)

 Take antibody binding sites, and join to fully humainsed strucutre.

Question 27

Affinity vs avidity

Answer 27

affinity is the strength of a single interaction between an antibody and its epitope

Avidity is the sum of the different affinities (some antibodies will be multimeric and will have several binding sites)