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Molecular Immunology
Lecture 1 – Introduction
Manipulation of the Immune System





Active immunisation (vaccination) – harmless pathogen/component to provoke an
immune response
...
g
...

The immune system can cause disease e
...
allergy
...
g
...


The Immune System

Cells of the Immune Response
Pluripotent stem cells -> Haemopoietic stem cells -> Myeloid cells + Lymphoid cells
Polymorphonuclear Granulocytes and Mast Cells





Neutrophils: produced in bone marrow, main phagocyte, short lived and fast moving
...

Basophils: stain blue with basic dye, similar to tissue mast cells in function
...


Mononuclear Phagocytes (myeloid)







Monocyte (blood) -> Macrophage (tissues)
Produced in the bone marrow
Phagocytic
Live for months/years
Present antigen to T helper cells

Antigen Presenting / Dendritic Cells (myeloid)



Present antigen to T cells
High levels of MHCII proteins

Lymphocytes (lymphoid)


NK cells: kills infected cells by recognising ‘altered self’, possible anti-tumour role as
well?

Phagocytic Receptors


Pattern Recognition Receptors (PRRs) recognise Pathogen Associated Molecular
Patterns (PAMPs)
...

Interferons: made by all cells in the body, interferes with viral replication
...


Adaptive Immunity
B Cells: secrete
antibody in the
humoral response,
involved in
extracellular bacteria
and secondary viral
infections
...


Immunoglobulin Classes
IgG: main class in serum/tissues, secondary response
...

IgA: secretions and mucosal surfaces
...

T Cell Immunity



T helper (CD4+ve): help B cells, activate macrophages and NK cells, help develop
CD8+ve cells
...


Cytokines






Small (~5-20kD) secreted proteins in communication
...

Interleukins (IL1-38) – usually made by T cells
...

Chemokines – chemotaxis
...

Th1: inflammatory, produce IL2, also produce IFNy and TNFa to activate
macrophages -> inflammation and phagocytosis
...


Immune System



Innate and adaptive immune system co-evolved
...


Lecture 2 – Innate Immunity, PRRs and PAMPs
Innate immunity is the older form of immunity, shared by plants, insects and mammals
...
It is always available which is especially
important in children between the time they lose maternal antibodies and are yet to
develop their own
...

Adaptive has ~4-6 day delay, whereas innate is crucial straightaway
...


Mechanical Barriers and Unfavourable Environments





Epithelia: shedding of top layer e
...
skin, airways
...

pH: too high in mouth and GI, and too low in stomach
...


Stages of Innate Immunity
1) Frontline Defence: specialised cells embedded in tissues, as well as soluble factors in
body fluids
...

3) Recognition
...

Inflammation






Causes redness, swelling, heat and pain
...

Dilation -> increase permeability and blood flow
...

However, inflammation is inhibited by the CNS as the CNS is physically protected by
bone, membranes, and fluid so there is no room for swelling
...


Pattern Recognition




Innate recognises relatively invariant structures
...

DAMPs (danger) are often critical for host cells e
...
tumours
...

Fragmentation of extracellular matrix components – release of proteases
...

Extracellular chromatin
...


PRRs






>1000 PAMP/DAMP ligands
...

PAMPs are exogenous ligands, DAMPs are endogenous ligands
...

Binding leads to cell activation or ligand uptake
...

Adaptive -> assembled in lymphocyte development
...

TLR Structure: TLR3
extracellular domain has a
horseshoe shape formed
by Leu rich repeats; the
inner surface has a B-sheet
structure and forms a
ligand binding domain
...

One of the most potent activators of the immune
system
...

LD50 in mice is ~150mg
...

Effects mediate by secreted cytokines
...

2) This allows binding to TLR-4 – more sensitive to low LPS concentrations
...

TLR4 Signalling
1)
2)
3)
4)

LPS binding induces the formation of TLR multidimers
...

Protein kinases e
...
IRAK, amplify the signal
...


MyD88 Pathway




Activates kinases such as IRAK
...
g
...

Causes immediate effects
...

Transcription of INFs a,b, and other genes
...


NFkB




Shared pathway between plant and animals
...

Constitutively active in cancers – may protect against apoptosis?

Lecture 3 – Innate Immunity, Cytokines
G-Protein Coupled Receptors






N-formyl-peptide receptors (FPR) act as PRRs – 7 TM domains, 3 in humans (more in
mice)v coupled to Gai
...

Other ligands include: other pathogen derived N-formyl-peptides; MHV binding
peptide; Altered self e
...
prion peptide, Amyloid B (Alzheimer's); Host defence
proteins
...


Inflammatory Mediators






Small molecules – ROI and RNI (Reactive Oxygen/Nitrogen Intermediates)
...
g
...

Complement factors – C3a and C5a
...

Purines e
...
ATP
...

Produced by many cell types
...

Local or systemic, autocrine or paracrine
...

Stimulatory or inhibitory (the same cytokines can vary at different pathway stages)
...

Receptors include: TNF receptors; Type I family; Type 2 family (mainly IFNs); Ig
superfamily; TGFB family; chemokine receptors
...

TM protein which is released via proteolysis to activate a response (LPS is a potent
stimulator)
...

Multiple types of receptor (TNFRIA most common)
...

Coordinates adaptive response in lymphocytes
...


TNFR1 Receptor
1) TNF trimer binds 3 TNFRs, bringing them together and enabling activation
...

3) OR signalling via TRAF
...

TRADD/FADD -> caspase and JNK -> apoptosis
...

2) Microthrombus – linked between inflammation and coronary thrombosis
...

TNF Sytemic Effects




TNF < 1ug/ml – activates receptors all over the body
...
5oC is also
optimum for T/B cell activation
...


TNF Sepsis





TNF > 1mg/ml – septic shock
...

Disseminated thrombus formation -> cardiac infarction and organ damage
...


Anti-TNF Therapy





Use antibodies against TNFa for rheumatoid arthritis and psoriasis (though not for
septic shock as there is just too much TNFa as well as other molecules)
...

Very expensive but very effective
...


Cytokines and T Cell Development
Th1 Cytokines
1) Viral/bacterial infection activates T cells
...


3) Viruses induce IFNa/B and bacteria induce IL-12/-18 and IFNy
...

Th17 Cytokines
1) Activated by bacterial and fungal infections at epithelial surfaces
...

3) Induces neutrophil recruitment and Th17 production – also implicated in
autoimmune diseases such as multiple sclerosis and rheumatoid arthritis
...

2) Th2 cells produce IL-4, -10, and -13
...

Intracellular Viruses
Type I INFs (a/B)



Intracellular TLRs 3,7,8, and 9 on endosomes, activated by dsRNA
...


INFg







Type II INF
...

Anti-viral response by a restricted range of cells
...

Activation of macrophages in the Th1 response
...


INF Signalling by JAK-STAT
1) INFa/B binds heterodymeric Type I receptors
...

3) STAT: signal transducer and activator of transcription
...

Protein kinase R/P1 kinase: uprated transcription activated by viral dsRNA, inhibits
all protein synthesis in the cells
...

Increased killing of infected cells via increased MHCI expression, so cytotoxic T cell
can recognise
...


Unfolded Protein Response





Innate immunity against viruses – stress response to ER protein accumulation
...

High levels of proteins – apoptosis, translation inhibition
...


Immunity against Intracellular Bacteria




Mtb controlled in 90% of infections – evolved to live with the host, however if
immunocompromised it can become fatal e
...
HIV
...

However, macrophage still realises it is infected so forms a granuloma via IFNy and
TNF production through TLR1/2(?)
...

Core of infected macrophages surrounded by uninflected macrophages, most of
them multinucleate Langhans giant cells
...

However it does provide a safe environment for Mtb and enables spread
...

~50 split into 4 groups, dependent on the spacing of Nter Cysteine
...






Induced and constitutively expressed
...

Different patterns in Th1 and Th2 cell development
...

Gradients of chemokines form on cell surfaces and the extracellular matrix
...


Chemokine Redundancy




Multiple chemokines bind to any one receptor – binding pattern due to gene
duplication events
...

Viruses make replica chemokines and receptors to mimic binding
...

Different tissues have different macrophages e
...
Kupffer cells
...

Express wide variety of PRRs
...

Produce pro-inflammatory mediators (TNF, IL-12,6,1b)
...


M1/M2 Macrophages





Analogy with Th1 and Th2 cells
...

Balance critical for inflammatory control – M1 act earlier and are proinflammatory,
whilst M2 act later and remodel tissue whilst also be anti-inflammatory
...


Innate Immunity and Cancer




Solid tumours release cytokines which attract macrophages and mast cells
...

However, tumours attract more M2 macrophages which aid progression via
angiogenesis, GF secretion, and metastasis
...

Huge numbers released from bone marrow
...

Found in large numbers in pus
...

Release antimicrobrials
...

Entrap microbes in DNA nets
...

Undergo successively tighter binding to capillary endothelial cells, then migrate
along chemoattractant trails immobilised in glycoproteins
...

Larger than lymphocytes
...

Cytotoxicity – no prior immunisation required (unlike CD8+ve cells)
...

Stabilises the virus before adaptive immunity acts, however it cannot remove the
virus
...


NK Cell Receptors




Controlled by stimulators and inhibitory receptors
...

Inhibitory receptors include killer Ig-like receptors, whose ligands include unaltered
MHCI
...

Essential? Conserved over 500 years, no reported absence in humans
...

Primary role in innate and adaptive immunity
...

Involved in many disease processes
...

Basophils (1%) and eosinophils (2-3%) circulate in the blood with short half-lives,
mast cells are only found in the tissues
...

Mast cell derived cytokines promote a Th2 response and can cause B cell class
switching to IgE
...


Lecture 5 – Innate Immunity
Complement



Complement cascade: complex system of plasma protein, induce cytotoxicity,
inflammation, and opsonisation
...


Complement Cascade





Activated by antibody complexes, foreign surfaces, and mannose residues
...

Components found in high concentrations in plasma and other fluids
...


Classical Pathway of Complement Activation
1) Heterotrimeric PRR C1 binds Fc of multimeric IgG1-3 or IgM, or directly to
pathogens, activating C1r and C1s
...

3) C4b binds C2, which is cleaved by C1s to C2a and C2b (serine protease), forming the
C4b2b complex
...
C3a is released
...

Downstream steps to the classical pathway
...

This reacts with hydroxyl groups, which takes precedence over amino acid groups
due the close proximity of a His residue
...


Alternative Pathway of Complement Activation
1) Intact C3 undergoes a slow rate of reaction with water at the thioester, forming
C3(H2O)
...


3) C3(H2o)Bb is a transient C3 convertase, producing more C3b – if there is enough
carbohydrate, C3b thioester will react to form a stable C3bBb, if not then it will bind
the microbial surface
...

Regulation of the Alternative Pathway




Host cells (and some pathogens) have complement regulators such as decay
accelerating factor (DAF) on their surfaces
...

Binding to pathogen factors e
...
P, reduces the rate of inactivation
...

There are complement receptors on B cells, macrophages, and neutrophils, which
cross-link via deposited opsonins to stimulate phagocytosis
...

C5b binds C6 to form a soluble complex
...

C8 binds, C9 binds and polymerises to form a pore in the lipid bilayer
...


Inflammation




C3a and C5a bind GPCR on neutrophils, monocytes etc
...

Elevated in sepsis and chronic inflammatory conditions such as rheumatoid arthritis
...

Commonly triggered by allergens e
...
house dust mites
...


BAD
C3a and C5a increased the lung fluids of
asthmatics
...


GOOD
C5 deficient mice more likely to develop
asthma
...


Blocking C3a and C5a receptors in models
can attenuate from the disease
...

Directing the Adaptive Immune Response



A foreign substance with few PAMPs but causes complement activation is opsonised
and activates macrophages via C5a – adaptive immunity is not switched on
...


Ebola Virus




Lethal in 50% of cases
...

Virus not airborne, spread via contact
...

Viral VP24 inhibits STAT1 translocation to the nucleus
...

IFN production decreased and multiple IFN-dependent events are blocked
...

Increased apoptosis of NK and T cells by Fas/Fas-L cascade – reduced ability to clear
virus
...


Lecture 6 – Adaptive Immunity
Danger Hypothesis – Matzinger, 1994



Body responds to ‘danger signals’ generated endogenously by injured tissue, not
foreign matter
...


Key Features of Adaptive Immunity





Primary contact – innate and weak adaptive immunity
...

Immune system eliminates infections via effector functions
...


Clonal Selection Hypothesis



During infection, B cell with correct receptor divides and differentiates in plasma
cells and memory cells
...


Organisation of Lymphoid Tissue



Primary –lymphocytes reach maturity (bone marrow and thymus; antigen
independent)
...


Activation of Adaptive Immunity
1) Lymph drains from infected tissue, containing antigen presenting dendritic cells
...

3) Produces plasma cells which enter tissues
...

Antibodies (anti-foreign bodies) – produced in response
...

Antibodies (immunoglobulins exist in two forms):
1) Integral membrane proteins on B cells (antigen receptors)
...


Basic Antibody Structure
Fab (recognition) – variable, bind antigen specifically
...

Hinge – enables epitope binding on different distances
...
They
then used an SDS Page gel to calculate the molecular weights
...

 Pepsin – Fab2 and Fc
Immunoglobulin Classes




Heavy – G, M, A, D, and E
Light – kappa and lambda
Mainly identified using antibodies

Discoveries from Myeloma Proteins
1) Constant and Variable Regions: V bind antigen and differs between antibodies with
different specificities
...

2) Homologous Domains: each domain ~110 amino acids with an intrachain S-S bridge
that folds to form compact globular domains
...

Folding pattern known as Immunoglobulin Fold (via X-ray crystallography of Fab and
Fc fragments)
...
Loops at Nter associated with antigen recognition
...
Each ~7-12 amino acids
...
Also called Complementary Determining Regions (CDRs)
...
Other framework regions maintain overall domain folding
...

Found in all members of the Immunoglobulin Gene Superfamily – largest family in
humans (756 members)
...

Members involved in recognition, binding and adhesion
...

Very stable and variability allows evolution
...

Individually weak (specific and high affinity) but many complementary residues
...

All immunoglobulin classes, but mostly IgM/D
...


T Cell Receptor






Only on membranes
...

CD8+ve – specifically kills infected hosts
...

CD+ve regulatory – suppresses immune reponses
...

T cell – intracellular, ‘cell associated’ and processed antigen

Major Histocompatibility Proteins




MHCI: Expressed by all nucleated cells and recognised by CD8+ve cells; after a viral
infection, proteins are broken down endogenously in the cytosol and the resulting
peptides are transported to the ER where they binds MHCI  cell surface
...


The T Cell Receptor (TCR)
Each V region has 3 hypervariable regions/CDRs
...

TCR recognises complexes of antigen + self MHC
...

TCR Complex



Requires association with additional proteins for signal transmission and cell surface
trafficking
...


Immunological Diversity




~1014 antibody receptors and ~1018 T cell receptors in humans, but only ~30,000
genes in genome
...

Diversity: somatic recombination and mutation of a limited number of inherited
gene segments, which make up the V regions
...

Susumu Tonnegawa (1976): immunoglobulin genes rearranged during B cell development
...

Gel electrophoresis
...

Found to be far apart in the embryo cell, but close together in the myeloma cell –
germline pattern found in all cells except B cells, where genes had moved during
differentiation
...

Light V regions encoded by 2 DNA segments
 V (90%) and J (joining, 10%)
Heavy V regions encoded by 3 DNA segments
 VH, DH (diversity), and JH
 Cu (IgM heavy) always expressed first in the immune response
...

In germline DNA, a V gene is splice next to a J gene with intervening DNA excising via
RNA processing, in order to form B cell DNA
...


Mechanism of Recombination




Lymphocyte specific recombinases
...

RSSs directly adjacent to coding sequence of V, D or J
...
g
...

Products of Rag-1/2 genes, which are specialised endonucleases expressed in
developing lymphocytes – mutations cause severe combined immunodeficiency
(scid)
...


The RSSs flanking the genes to be recombined are brought together
...

Mechanisms of Diversity
1) Multiple copies of each V region gene segments (V, D, J)
2) Any heavy x light chain combination - ~37x106
3) Recombination is imprecise  junctional diversity
a
...
TdT randomly adds NTs to V-D-J joins
c
...

Ig Gene Expression and B Cell Differentiation (Antigen Dependent)
1)
2)
3)
4)

HC rearrangement (D-J then V-DJ, TdT adds up to 12 NTs (N region))  uHC
LC rearrangement (V-J)  expresses membrane IgM
Selection against self
Secondary lymphoid tissue  B cell expresses IgM or IgM + IgD

Somatic Hypermutation





Occurs throughout rearranged V regions, but in mature B cells they appear to be
clustered in CDRs
...

Activation – induced cytokine deaminases (AID) introduce mutations
...


Class Switching


DNA recombination between
switch regions – intervening DNA
is lost and it is irreversible
...
This allows simultaneous membrane expression of two classes, and it also
reversible
...

As B cells become more active, the secreted form predominates
...


Diversity of T Cell Receptor Genes
1)
2)
3)
4)
5)

Multiple copies of each V region gene segment
...

Somatic mutation is avoided so that they can recognise self MHC
...







Fewer V regions, junctional variability may compensate
...

Less diverse, recognise a broader antigen range
...


Lecture 9 – MHC Proteins






Encoded by genes of the Major Histocompatibility Complex on chromosome 6
(humans)
...

Very polymorphic e
...
>1400 alleles of HLA-B locus, which may differ by up to 20
amino acids
...

T lymphocytes can only recognise antigen in context of self-MHC molecules –
discovered using experiments with virally infected cells and inbred mice
...

MHCII – certain leucocytes; present peptides from exogenous proteins to CD4 cells
...

MHCII has a polymorphic a and B chain
...

Membrane distal domains bind peptide and contain polymorphisms
...
The Nter and Cter of the proteins bind to
invariant pockets at the ends of the groove, and 2-3 ‘anchor residues’ bind to the specificity
pockets formed by polymorphic residues
...
The peptide backbone interacts with invariant
residues whilst side chains interact with specificity pockets at the bases of the peptide
binding groove
...

X-Ray Crystallography
1) MHC binds peptide
...

3) MHC have a broad specificity for peptides (degenerate), and the bound peptide
dissociates very slowly
...

Peptides transported to ER via an ATP-hydrolysis driven transporter
...


T Cell Activation





TCR must associate with other cell surface molecules (CD3 + ζ chain) for signalling
(TCR complex)
...

Binding of MHCII invariant region to TCR phosphorylates ITAMs of TCR via receptor
associated kinases
...


MHC Genes




Gene loci very polymorphic with multiple alleles, each denoted by a different
number e
...
HLA-B143
...

Polymorphisms clustered in distal peptide binding domains
...

MHC Protein Function







Graft rejection
AP to T cells – activation
Recognition of self/nonself (NK cell KIR killing inhibitory receptors)
Association with certain autoimmune diseases
Development of T cell repertoire/tolerance in thymus
Influence mate choice?

Thymic Selection

Lecture 10 – Immunoglobulin Effector Functions
B Cell Responses





Antigen independent differentiation in bone marrow
...

Once activated, they differentiate into plasma and memory cells in the secondary
lymphoid tissue
...


IgG









Monomeric molecular weight of
150,000
...

Subclasses IgG1, 2, 3, and 4 – differ
mainly in length and number of
disulphides at hinge
...

Binds Fc receptors on phagocytes and
NK cells
...

Long serum half-life (FcRn)
...


IgM








Pentamer – 5 antibody subunits + J chain
...

No defined hinge – instead two extra subunits
...

Can activate complement very efficiently
...


Primary and Secondary Responses



Isotope switching  IgM  IgG, A, or E
Helper T cell cytokines usually required
...

IgA1 and IgA2 subclasses (primates)
...

Secretory IgA is an IgA dimer + J chain + secretory component
...

Rapid catabolism
...

Binds Fc receptors on phagocytes
...
Mucosal lymphoid tissue produces 5 IgA’s per day
– main role?
IgD





Monomer in molecular weight of
184,000
...

Present as an antigen receptor on many
B cells, as well as IgM
...


IgE






Molecular weight of 190,000
...

Trace in serum (0
...

Binds to high affinity FcR on mast cells and basophils to release histamine
...


Biological Role of Immunoglobulins








Label pathogens – elimination
...

Neutralise toxins (IgG, A)
...

Prevent pathogen binding to the host (IgG, A)
...

Form ‘immune complexes’ with soluble antigen
...

Activate complement (IgM, G)
...


Complement



Discovered as a heat sensitive factor in serum which complemented the activity of
antibody in losing foreign red blood cells
...


Many have protease activity and fragments can be generated with biological activity e
...
C3
--> C3a + C3b
Roles of Complement
1) Clearance of immune complexes to defend against bacteria and viruses
...

Pathways of Complement Activation
1) Classical
a
...
C1r2s2 (requires calcium ions)
c
...
This requires Ag/Ab2, can be C1, 2, or 4
2) Alternative
3) MB-Lectin Pathway
Central event in all of these is C3 cleavage into C3a and C3b
...

IgM is much more potent activator than IgG (IgG3 > 1 > 2)
...

Complement activation results in
results in inflammation, leukocyte
activation (e
...
for chemotaxis),
opsonisation, and lysis of foreign cells
...

2) Activation of immune system (C5a and C3a) – via chemoattractants
“anaphylatoxins”
...

Most function as part of a multi-subunit complex
...

Activated by FcR bound antibodies binding antigen – involves immunoreceptor
tyrosine activation motifs (ITAMs)
...

Different isoforms of FcRs (more for IgG) – differ in cytoplasmic and membrane
spanning regions
...

Via IgG and FcRIII (CD16)
...

Binds IgG with high affinity
...

Recently shown to neutralise virus infection in vivo
...

Mast cells secrete inflammatory mediators and cytokines
...


Cytokines can induce or inhibit IgG isotope expression
...

Thymus independent antigens – e
...
bacterial polysaccharides; induce a more rapid
response and IgM production, memory cells not generated
...
g
...

T cell subsets and their cytokines can influence antibody (sub)class produced in the
humoral response
...

 Movement of T cells and initial interactions to APC are mediated via adhesion
molecules e
...
integrin
...

 Interaction between co-stimulator pairs deliver the second signal
...

IL-2 acts in an autocrine fashion on T helper cells and is required for cytotoxic T cell
activation
...


Drug TGN1412




Developed by Tegenero for treating autoimmune disease and leukaemia
...

Though most antibodies block activity, this one binds CD28 on T cells, stimulating
them and causing a cytokines storm
...

CD4 helper cells (Th1, 2, 17, TFH)
...

 Activate B cells for class switching and somatic hyper mutation
...

CD4 Regulatory Cells
 Suppress immune responses
...




CD8 Cells
 Kill infected hosts
...

These activate macrophages, which induce inflammation
...

Induce B cells to make IgG1 and IgG3 (opsonising antibodies)
...


IL-4 (signal 3)





Th2 cells activated to make IL-4,5,6,10 and 13
...

Helminth infections and allergy
...


TGFB, IL-6 (signal 3)






Activates Th17 CD4 cells to make IL-17 and IL-22
...

Proinflammatory, especially at the mucosal surfaces
...

Important in fungal and extracellular bacterial infections, maybe a role in
autoimmune disease?

IL-6 (signal 3)





Activate TFHCD4 cells (follicular helpers)
...

Help B cells differentiate and promote somatic hyper mutation
...


Selective production of different CD4 effector subsets can have profound consequences for
the type of immune response produced
...

Once activated, they bind specifically to infected target cells and induce apoptosis:
a) Proteases (granzymes) from CD8 cell enter target via perforin channel
...

Can also produce some cytokines e
...
IFNy and TNFa
...

IFNy and TNFa can synergise in activating macrophages and killing some target cells
by inducing apoptosis
...

Bug negative correlation between helminth infections and allergic disease
...

Worms + allergens -> naïve CD4 produces IL-4 (via dendritic cells and mast cells) ->
Th2 -> IL-4 stimulates IgE and decreased killing
...

Human immune system and ‘old friends’ co-evolved
...

May also explain the tide in autoimmune disease (Th1/17 – driven)
...

Diverse (>109 specificities)
...

Domain structure – stable, facilitates engineering
...

Effector properties – useful in some techniques and therapeutics
...
e
...


Antibodies as Molecular Probes




Antibodies covalently labelled with an easily detectable tag
...

Fluorescent; enzyme -> coloured product (ELISA); radioisotope; gold particles; latex
beads; sepharose
...


Generating Antibodies







Immunogenicity – ability to induce an immune response
...

Molecular size - <1000 Daltons (carrier proteins)
...

Ability to provoke T cell responses – genotype of recipient
...


Epitopes






Recognised by antibodies on an antigen
...

Discontinuous – non-adjacent (conformational)
...

Antibodies can bind monovalently or multivalently to repeated epitopes
...

Mixture of antibodies specific to different epitopes
...


Polyclonal Antisera




Advantages – cheap, robust (may recognise partially denatured antigen), form
immune complexes well
...

Uses:
1) Secondary antibodies for immunoassays
...
g
...

3) Identification of gene products e
...
Dystrophin gene in DMD
...

Monoclonal Antibodies






Derived from single B cells
...

Advantages – highly specific, can be standardised, pure antigen not needed for
immunisation
...

Diagnostics – detect and quantitate diagnostically important molecules in clinical
samples e
...
pregnancy/fertility test, Down's syndrome; also to serotype pathogens
(closely related strains)
...

Identify cell types e
...
T cells -> CD4 and 8 -> stage of differentiation
...

Biochemical analysis, purification
...

Monoclonal antibody technology not readily applicable for generating human
antibodies
...
g
...
Antibody engineering can be used to make antibody chimeras (mouse V
regions, which are still immunogenic, to human C regions) and ‘humanised’ antibodies
(human framework regions and mouse CDR regions aka CDR grafting, and is facilitated by
immunoglobulin domain structure – though my lose affinity/specificity and is also time
consuming)
...

2) Amplify Fab or Fc cDNA by PCR
...
g
...

4) Screen antibody phage display library vs solid phase antigen ‘panning’
...

2) Construct fusion protein of V region with a bacteriophage coat protein
...

4) Select phage with desired V regions by specific binding to antigen (panning)
...

Types of Antibody Gene Libraries






Immune - immunised human sources
...

Semi-synthetic - >109 members, human Ig V region genes (germline) plus randomly
generated CDR3 regions
...

Either selected on phage or using cell-free expression systems e
...
HuCal Gold
(Morphosys)
...


2) TRANSGENIC Mice expressing human immunoglobulin genes, which are introduced
using yeast artificial chromosomes (YACS) – mouse antibody genes replaced by
humans (xenomouse), immunised to generate human antibodies by conventional
monoclonal or phage display techniques
...

Nanobodies consist of one variable domain only from a heavy chain only antibody
...

Highly potent, robust and stable
...

Ease of manufacture and multiple administrating routes into the body
...

Improving Antibodies for Therapy
You can map effector sites on antibodies and if done precisely, may be used to generate
‘designer antibodies’ for use in therapy
...
You can
increase half by additions of polyethylene glycol (PRGylation)
...
g
...

Fv – paired variable regions
...

Increase valency – diabodies, tribodies
...
g
...

Immunotoxin – retain antigen specificity, tag or replace Fc with toxin e
...
Diphtheria, ricin
...
g
...
g
...

Cancer/chronic disease – antibodies as magic bullets to target tumour cells e
...
AntiCD25 antibodies which recognise leucocytes and activates complement and ADCC,
used in leukaemias and lymphomas; e
...
Anti-Her2 antibodies (herceptin) which
recognise Her2, a receptor tyrosine kinase which is highly expressed in ~25% breast
cancers
...


Antibodies to Modulate the Immune Response







Depletion of leucocytes e
...
Antibodies to CD25, CD3 and CD4
...

Blocking of cytokines, their receptors, and soluble mediators
...

Allergy (to IgE)
...


Co-Stimulatory and Co-Inhibitory Molecules







B7 interaction with CD28 induces stimulation
...

CTLA-4 induced on activated T-cells and expressed by TREGS; it has a higher affinity
for B7 – inhibition of T-cell
...

CTLA-4 and PD-1 acts as immune checkpoints
...


More than360 antibodies are now used as therapeutics e
...
CAMPATH in B cell chronic
lymphocytic leukaemia, and Herceptin in breast cancer

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