Download 1st seminar Ag, Ig, monoclonal 2016

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ANTIGEN, ANTIBODY
Topics:
-
immunogenicity, tolerogenicity
Term of antigen
epitops (B-, T-cell epitops)
hapten
carrier
antigen presentation
T independent antigens
superantigens
-
immunoglobulins
structure of the antibodies
Isotypes
antibody functions
policlonal and monoclonal antibodies
passive immunisation
humanised antibodies
antibodies in practice (first part)
practice:
- the basics of the production of monoclonal antibodies (hybridome cell lines)
- cell counting
- fast recloning of the cells
- antibody purification by affinity column
You should know this already…
ANTIGEN
TERM of the IMMUNOLOGY
TERM of the ADAPTIVE IMMUNITY:
• In connection with B-, T-cells which have ANTIGEN RECEPTORS
• In connection with ANTIBODIES which recognise ANTIGENS
Any chemical structure
(But they should be able to form secondary bonds with proteins, as the antibodies and the antigen
receptors are proteins.)
Soluble or corpuscle
Simple or complex
Originated from the body or comes from outside
Genetically self or non-self
Natural or artificial
You should know this already…
DEPHINITIONS
• Antigen (Ag) – Any kind of substance which can be
recognised specifically (with antigen receptors) by the
immune system
• Antigenicity – capability of an antigen to bind specifically
with the antigen receptors, or antibodies of the adaptive
immunity:
– immunogenicity - capability of an antigen to induce
an (adaptive) immune response
– tolerogenicity - capability to induce immunological
tolerance, specific immune”non-responsiveness”
(usually in the case of self-antigens)
You should know this already…
FACTORS INFLUENCING
IMMUNOGENICITY I.
From the aspect of the body:
• Genetics (self/non-self)
– species (evolutionarily nonconserved molecules)
– individual differences (e.g. MHC polymorphism – see later)
• Age
– newborn – less reactive immune system
– elderly – no new lymphocytes
• Physiological condition (e.g. immunodeficiencies, starvation)
• Immune privilege – special, non immunogenic site of the body:
brain, eyes, placenta, testicles
You should know this already…
FACTORS INFLUENCING
IMMUNOGENICITY II.
From the aspect of the antigen:
• Physical-chemical properties of the Ag
–
–
–
–
size/complexity (bigger  more epitopes, role of carrier)
corpuscular (cell, colloid) or soluble
denatured or native (different epitopes!)
degradability (by APCs)
You should know this already…
FACTORS INFLUENCING
IMMUNOGENICITY III.
From the aspect of vaccination:
• Dose
• Route
Általában: intradermal/subcutan > intravenous > oral > intranasal
(the order depends from the antigen also – e.g. oral vaccine against polio virus)
• Adjuvants
They can modify (enhance) the efficacy of the vaccine, but they are
not immunogenin by themselves
e.g. aluminum hydroxide precipitates, mineral oils, Freund-adjuvant,
TLR ligands, cytokines (IL-12, IL-2)
COMPLEX EFFECT:
• depot effect  increased presence of the antigen
• activation of the natural immunity and its accessory cells
(e.g. APC)
(animated slide)
You should know this already…
The antigen recognition
of the B- and T-cells
APC
antigen-specific
antibodies
Antigen
MHC
BCR
TCR
(membrane Ig)
T
B
• B cells and antibodies can recognise ”native” (unmodified)
antigens
• T cells recognise processed antigens
(animated slide)
Antigen: Any kind of substance
which can be recognised specifically
(with antigen receptors) by the
immune system
Antigén
Antigen determinants (epitops):
Those parts of the antigen which
the antibodies or antigen receptors
directly bind (with secondary
bonding forces / intermolecular
forces)
You should know this already…
B cell epitope
T cell epitope
(recognized by B cells)
(recognized by T cells)
• proteins
polysaccharides
lipids
DNA
steroids
etc. (many artificial
molecules)
• protein derived peptides
• cell or matrix
associated or soluble
(8-10 or 10-20 amino acids)
requires processing by APC and
presenting on MHC molecules
• some other small
molecule: e.g. glycolipids,
lipopeptides
requires processing by APC and
presenting on MHC like molecules
as CD1 molecules
(animated slide)
The structures of the antigen can be
principally divided to epitopes and ”carrier”
•
•
antibody 1.
The ”carrier” could be considered as a part of
the antigen without the epitopes
Carriers have a real meaning in vaccine
production (especially in the case of haptens)
”carrier” (2.)
antibody 2.
epitop 1.
antigen
epitop 2.
”carrier” (1.)
The basic types of the antigen determinants
linear determinant
conformational determinant
(TCR, BCR, Ig)
(BCR, Ig)
conformational
determinants
Ab2
Ab1
normal, accessible (cell surface)
determinants
cleavage
denaturation
new/(neoantigenic)
determinants
(neoepitopes)
Western Blot  denaturing environment
conformational/linear
could destroy the conformational antigen
determinants
determinats and can reveal cryptic antigen
determinants (either linear or conformational)
cryptic
determinants
You should know this already…
Main types of antigen determinants
Linear antigen determinants usually not affected by denaturation:
ideal targets in Western-Blotting
(animated slide)
Antigen presentation – typical examples of the
linear neoepitopes for T cells
CD8+
cytotoxic
T cell
CD4+
helper
T cell
exogenous proteins
MHC I
MHC II
antigen
presenting cell
(APC)
endogenous proteins
MHC molecules can be considered as chaperones which stabilise the antigen derived peptides
into an elongated, linear conformation.
Epitope mapping (generally for linear epitopes)
Protein antigen (amino acid chain)
PepScan:
Cleaved, purified antigen fragments are
immobilised on a surface. Antigen specific antibodies can
choose the appropriate fragment with the epitope.
peptides
T cell epitopes: The peptide fragments can be given to
antigen presenting cells. The isolated peptide with the T
cell antigen determinant will activate the T cells in the cell
culture.
Ala-Scan:
After the PepScan, the sequence of the
reacting peptides can be modified (peptide synthesis)
systematically. (Usually the amino acids are changed to
alanine, one-by-one)
If an amino acid change destroys the recognition, that
amino acid should be a ”contact” amino acid.
A T G L R N I P S
V A G L R N I P S
V T A L R N I P S
V T G A R N I P S
…
Genetical methods:
Site directed mutagenesis
Phage display library
V T G L R N I P S
etc.
You should know this already…
Thymus independent antigens (TI antigens)
Some antigen can effectively activate the B cells without the presence of helper T cells
T INDEPENDENT ANTIGEN 1
TI-1
T INDEPENDENT ANTIGEN 2
TI-2
B CELL
The antigen mediates
signalling through the BCR
and some other cell surface
receptor (e.g. LPS receptor)
simultaneously
Dense, repetitive epitops (e.g.
polysaccharides on the bacterial
cellwall) mediate efficient BCR
crosslinking
B CELL ACTIVATION
Additional activating signal next to
the antigen receptor signal
extensive receptor
crosslinking/aggregation
ANTIGEN, CARRIER, IMMUNOGENITY, VACCINES
• Bacterial cell surface glycoproteins, glycolipids,
polysaccharides are efficient targets for the immune
recognition.
• They have extreme variability between bacterial strains
Polysaccharide antigens
from different
Pneumococcus
strains
Polysaccharides  no Ag presentation for T cells:
• No isotype switching
• No affinity maturation
• No B cell memory
Short term, ineffective
(low affinity) IgM production
ANTIGEN, CARRIER, IMMUNOGENITY, VACCINES
Example:
Prevenar - pneumococcus vaccine
Polysaccharides can be conjugated to a protein antigen carrier. In this way a T
cell mediated B cellactivation can be achieved also.
The CRM197 protein are in use  modified diphteria toxin (toxoid)
(A nontoxic variant can be produced from the toxin with one amino acid change (Glu  Gly) that
is called ”toxoid”)
The toxoid is also immunogenic and can mediate anti-toxin antibody production
in the body.
Glu  Gly
toxin
toxoid
Polysaccharide antigens from
different Pneumococcus
strains
toxoid
toxoid
Complex vaccine antigen
ANTIGEN, CARRIER, IMMUNOGENITY, VACCINES
A B cell which recognise the polysaccharide epitops can
present the toxoid protein derived peptides for the T cells
polisaccharides
Peptide
from the
toxoid
toxoid
MHCII
BCR
TCR
Helper T cell
B cell
cytokines,
CD40-CD40L interaction
• Polisaccharide recognising memory B cells ( antibody producing effector cells)
• High affinity antibodies, isotype switching
Long term antibody production against different Pneumococcus strains
HAPTENS
Small molecules which are unable to induce immunresponse by
themselves
Immune
response
haptens
‒
(e.g. DNP: dinitrophenyl)
carrier + haptens
• Weak immunogenic antigens can be bound to carriers for more effective
immunogenicity
• It could happen in vivo with small inert or reactive molecules in the body
(medicaments) – hipersensitivity reactions (penicillin can bind to red blood cells)
+
Haptens and lymphocyte activation
free haptens
Carrier bound haptens
0
The first step of the antigen receptor associated Src-like tyrosine kinase
activation is the ”auto”phosphorilation. The receptors should be crosslinked or aggregated by the ligand.
The immun response against the carrier bound haptens
carrier+haptens
antibodies
carrier specific
hapten specific
carrier+hapten
specific
(conformational epitop)
SUPERANTIGENS
•
•
•
•
Microbial proteins. Microbial tool for immune evasion.
It can attach various Vβ type TCR to the MHC molecules on the APC.
It can mediate polyclonal T cell activation.
The binding and the activation is independent from the TCR specificity.
Normal
MHC+peptide
complex
Activation induced cell death
is mediated in the strongly
activated T cell
The superantigen
attaches the TCR
with the MHC
molecule outside
from the peptide
binding groove
It results ”cytokine storm” –
the body is weakened and the
produced regulating cytokines
supress the microbe specific
immune response also
B cell superantigens
• They bind to the antibody outside the antigen binding site: e.g. to the
constant domains in the Fc part
• The binding is independent from the antigen specificity of the antibody
• Superantigen bound antibodies unable to mediate effector functions
• They are able to mediate polyclonal B cell activation
• They can be used in the biotechnology to isolate or purify
antibodies
Bacterial and viral T- and B-cell
SUPERANTIGENS
Classification
Endogenic
Sources
1.Mouse mammary tomor virus (MMTV) (integrated retroviral superantigen in the genome)
2.Epstein-Barr virus associated superantigen (EBV infection can reactivate the endogenic retrovíral
superantigen (HERV-K18, ERKV-18)
Exogenic
.
1.Staphylococcal enterotoxins (SEs): A, B, C1 to C3, D, E, G to Q
2.Staphylococcal toxic shock syndrome toxin-1 (TSST-1)
3.Staphylococcal exfoliative toxins: exoliatin A, exfoliatin B
4.Staphylococcal enterotoxin-like toxins formed due to recombination within enterotoxin gene cluster:
U2, V
5.Streptococcal pyrogenic exotoxins (SPEs): A1 to A4, C, G to M
6.Streptococcal mitogenic exotoxins: SMEZ
7.Streptococcal superantigen :SSA
8.Yersinia pseudotuberculosis: Yersinia pseudotuberculosis-derived mitogen (YAM)
9.Mycoplasma species: Mycoplasma arthritidis-derived mitogen (MAM)
10.Cholera toxin: subunit A of cholera toxin
11.Prevotella intermedia*
12.Mycobacterium tuberculosis*
13.Viral superantigens: (a) Mouse leukemia virus
(b) IDDMK1222- Ppol-ENV-U3
(c) HIV-Nef
(d) Rabies virus-nucleoside protein
B cell
1.Staphylococcal protein A
2.Protein G
3.Protein Fv (PFv)
immunoglobulins (Ig)
structure and functions
Immunoglobulins
(glycoproteins)
• Belong to the gamma-globulin
electrophoretic mobility group
• Antigen specific proteins
• B cell surface antigen recognising
receptors  B cell receptors (BCR)
• Antibodies in the body fluids (blood
plasma, tear, saliva, etc…)
The structure of an immunoglobulin
• Light and heavy
chains
• Disulphide bridges
– interchain
– intrachain
• Antigen binding
parts and Fc part
• Hinge region (or
not)
• Domains
– VL & CL
– VH & CH1 - CH3
(CH4 e.g. IgM)
• Oligosaccharides
Mediation of
different effector
functions:
Antigen (epitop) binding
disulphide bridges
Fc receptor binding
complement activation
carbohydrate
CL
VL
CH1
VH
CH2
hinge region
CH3
Variable domain of an antibody
Immunoglobulin (like) domain:
~12kDa
Ribbon structure of IgG
The enzymatic cleavage of immunoglobulins
helped revealing the structure / function
relationships
Antigen
binding
Complement binding site
Binding to Fc receptors
Placental transfer
Immunoglobulin fragments:
structure/funkction relationships
Papain
• Fab (Fragment antigen binding)
– Ag binding (valence=1 monovalent)
– Specificity provided by the
VH and VL domains
VH
VL
• Fc (Fragment crystallizable)
– Effector functions
Fc
Fab
Immunoglobulin fragments:
structure/funkction relationships
Pepsin
• F(ab’)2
fragment
+
– Antigen binding
bivalent! valence=2
Fc
peptides
F(ab’)2
Some effector function is lost by the degradation of the Fc part, but
it can mediate agglutination and precipitation (next seminar)
The F(ab’)2 fragment is able…
•
Antigen recognition (binding)
•
Antigen precipitation (bivalent!)
• Blocking the active site of toxins or pathogen associated
molecules (neutralisation)
• Blocking the interactions between the host cells and the
pathogens (neutralisaton)
Without Fc region, there is no…
•
Cell mediated inflammation
•
Complement system mediated inflammation
• Efficien phagocytosis and subsequent antigen
presentation
You should know this already…
Antibody mediated effector functions
Fab
Fc
FcR
Fc
+ complement
system
Antigen binding
Model of the interaction between the antigene determinant
and the antigen bibding „pocket” of the antibody
Antigen – antibody interaction
Valence and avidity relationship
Valency: number of
connections between the
antigen and the antibody
Affinity: the „strength” of
the connection between
one epitope and one
antigene binding site
Avidity: the overall strength
of the interactions between
epitopes and binding sites
(like the teeth of the zipper
or velcro)
The valency of
antibodies
2
Free IgM pentamer
(”star” shape)
2
2
4
Antigen bound IgM
(”crab” shape)
10 or 12
tetra- or hexamer can be posible
The hinge region can have indirect role
in the efficient binding of the antigen
The hinge region helps in the efficient formation of
multivalent interactions
Repeated epitops are needed for
multivalent interactions with the antibody
Properties of antibody isotypes
NEUTRALISATION (mediated by the antigen binding)
The cells are damaged
The cells are protected by the neutralising antibodies
Antibodies of the memory immun response after immunisation or the passive
immunisation can mediate this process
Virus neutralisation
Protected
Host cell
INFECTED
Host cell
Neutralised
Virus
Virus
Virus
Vírus specific
neutralising
antibodies
„Virus receptors”
„Virus receptors”
Antibodies in the investigations of the cellular functions
• Blocking the receptor activity
• Receptor activation
Cross-linking of tyrozin kinase
receptors: activation
„Receptor neutralisation”
Fc receptor mediated functions
FcγR : γ  IgG binding
FcεR : ε  IgE binding
FcαR : α  IgA binding
and transfer Fc receptors
FcR
n
„neonatális” Fc receptor
Placental cells, endothelial
and epithelial cells, etc.
IgG transfer, IgG salvage
pIgR
poly Ig receptor
Epithelial cells
IgA, IgM transfer
The receptor activation/inhibition function of the
antibodies can be modified by the Fc receptors!
Antibodies with flexible hinge region can recruit Fc receptors
in the close proximity of the activated/inhibited receptors:
• Activating Fc receptors with ITAM motif
• Inhibitory Fc receptors with ITIM motif
antibody
RECEPTOR
inhibition
The usage of antibodies in the research can
mediate undesirable Fc receptor effects
Fc receptors can be present on the examined cells or
on the neighboring cells! The desirable cell functions
can be altered.
Aged antibody products (generally the proteins) tend to form
aggregates:
The aggregated antibody complexes can bind to
the Fc receptors very effectively!
Unwanted Fc receptor effects can be
avoided by the usage of….
Fab
F(ab’)2
…fragments!
Sometimes the lack of the Fc part cause the problems…next slides
The properties of the antibody isotypes
or hexamer
Antibody isotypes and functions
The significance of the Fc part
The different
isotypes show
different
distributions in the
body
pIgR: IgA transport, secretory IgA
SS
SS
ss
J
SS
S
S
ss
S
S
SS
SS
ss
SS
J
S
S
S
S
S
S
B
M
U
C
I
N
Epithelial
cell
pIgR internalizes
with the bound IgA
ss
SS
S
S
S
S
J
S
S
J
J
S
S
ss
SS
ss
S
S
S
S
SS
SS
ss
S
S
SS
J
J
S
S
SS
S
S
The pIgR bound
IgA transported
across the cell:
transcytosis
SS
The pIgR is cleaved by proteases, so
the IgA can detach from the cell
surface. The remaining receptor
fragment on the IgA is called: secretory
fragment
SS
Submucosal B cells (BALT, GALT)
produce dimeric (trimeric) IgA
“poly Ig receptors”
(pIgR) located at the
basolatheral surface of
the epithelial cells.
They can transport IgA
(and IgM) to the apical
surface
What is transported by pIgR (poli immunoglobulin receptor)?
Immunoglobulins with J-chain: (polimer) isotypes: IgA, IgM
(DO NOT MIX this J chain with the J region of Ig gene rearrangement (VJ/VDJ rearrangement)l!!!)
Where to transport?
• epithelial (e.g. gut- and lung) surfaces
• secretums (saliva, breastmilk)
The IgM can form
pentameric or hexameric
structures without the Jchain also, but this polimer
is not transported by pIgR.
Hexameric IgM can activate
the complement system
better than the pentameric
(an order of a magnitude).
IgG can form hexamers on antigenic
surfaces with dense repetitive epitopes
14 MARCH 2014 VOL 343 SCIENCE
• Superior complement activation
• No J-chain  no pIgR transport
The IgG can be transported by an other receptor
neonatal Fcg receptor (FcRn)
Human FcgRn
Human MHC
Class I
(structural similarity, but different function)
IgG can be transported also
FcRn or FcγRn
Transport of monomeric IgG and
albumin
expressing cell types:
- endothelial cells
- epitelial cells (digestive-, respiratory- and urogenital system)
- lots of cells of the immune system: e.g. professional APCs
The function of
FcRn
(rescue)
pinocitosis
FcRn binds the IgG with
high affinity in acidic
environment and rescue it
from the degradation
Transcytosis
IgG can be transported to various compartments in the body this way:
• blood (circulation)  tissues
• tissues  epithelial surfaces
(There are more IgG in the urogenital- or in the upper respiratory tract than IgA)
• mothernal circulation  phoetal circulation
The half life of IgG is rised by FcRn
FcRn overexpressing transgenic animals are excellent tools to
produce policlonal antibodies after the immunisation
Heavy chain antibody - hcIg
VHH fragment/nanobody
antigen
(lisozyme)
IgG: human, mouse, rat…
immunoglobulin new
antigen receptor
(IgNAR):
Cartilageous fishes,
sharks
hcIgG: Camelidae
camel, dromedary, llama
MONOCLONAL ANTIBODIES
and
POLYCLONAL ANTIBODIES
Antibody response
Ag
Immunserum
B cell set
Activated B cells
Policlonal
antibodies
Antibody producing
plasma cells
Ag
Antigen-specific
polyclonal antibodies
(the products of different B cell clones!)
The blood plasma of the immunised animals is a simple source for
antigen-specific antibodies.
You should immunise more animals for prolonged usage of the
antibodies
- The standardisation of the antibodies are difficult
- The amount of the antibodies are limited
A proliferating immortal antigen-specific B cell would provide infinite
source of antigen-specific antibodies.
Monoclonal antibody production
Georges J.F. Köhler, Cesar Milstein (Nobel prize 1984)
Antigen
Policlonal antibodies
Monoclonal antibodies
Monoclonal antibodies
The products of one B cell clone
Homogenous (antigen specificity, affinity, isotype…)
They can be produced in large quantity, and similar quality for
extended period of time (principally infinite)
It needs the production of proliferating, immortal
antigen specific B cell
„immortalisation”
?
- can happen „in vivo” in pathologic conditions
e.g. myeloma multiplex: malignant proliferation of a B cell clone  large quantity
of a monolonal antibody in the circulation
Myeloma multiplex
normál serum
patient’s serum
Possibilities?
Transformation/immortalisation by viruses
e.g. EBV – can transform human B cells
Drawbacks:
- The selection of the antigen specific clone is difficult
- The virus can appear in the antibody product as contamination
Possibilities?
Fusion with an immortal cell line
Requirements for the fusion partner:
- it should not produce it’s own antibodies
- the immortal status should be maintained in the hybrid
- it should not influence the antibody production of the
specific B cell
- selectability: some easy way to get rid of the aspecific
unnecessary cells
There are few good fusion partner cell line:
Mouse:
Human:
Sp2/0-Ag14
(SP2 cell line)
no good general fusion partner exists!
heteromyelomes, triomes can be used:
(The fusion partner could be a hybrid of 2-3 cell type by own.
Sometimes mice-human hybrid cells)
CB-F7, K6H6B5, H7NS, NAT-30, HO-323, A4H12
Fusion possibilities?
Micromanipulation
(needs expensive complicated hardware)
„FuCell”
Micromanipulation
antigen
B cell
fluorescent or paramagnetic
label
- The separation of the antigen specific B cells are mediated
by the antigen themself
- The B cells are placed in the vicinity of the fusion partner
- The fusion is mediated by electrical pulse
- As the B cell is antigen specific, there is no need for
multistep selection - the requirements of the fusion partner
are not so strict
The process of the fusion in the instrument
The production of the monoclonal antibodies
Generally: Hybridome production, cell fusion with mouse SP2 cell line
Hybridoma technology
 Short living antigen specific immune cells (B cells) are fused with immortal proliferating tumour
cells. Stable, proliferating, antigene specific antibody producing hybridome cells can be separated with
a careful multistep selection.
 The end products are specific antibody producing hybridome clones, and monoclonal antibodies.
Monoclonal antibody production in draft
immunisation
myeloma
antigen specific
activated B cells
spleen
- Immunisation of a laboratory animal
- Isolation of a lymphoid organ (e.g. spleen)
with antigen specific B cells
serum,
policlonal
antibodies
fusion
selection
- Fusion of the cells with the immortal
tumour cells
Antibodyselection
- Multistep selection of the specific antibody
producing hybridomes
The hybridome cells are continuously
producing the antigen specific antibodies
into the cell culture medium
(1.)
(2.)
Recloning the antibody-producing
clones
freezing
In vitro cultures
(animated slide)
Cell fusion
spleen cells
myelome cells (SP2)
PEG, Sendai virus, micromanipulation
Dying within days
Dividing (proliferating) cells
1st main selection step: Discarding the unnecessary myelome cells
spleen cells
HGPRT+ TK+
myelome cells (SP2)
HGPRT- TK-
drug sensitive
(„HAT” selection)
HGPRT+ TK+
HGPRT- TK-
The biochemistry of the „HAT” selection
Hypoxanthine, Aminopterin, Thymidine containing medium
nucleotide precursors
enzyme inhibitor
The „HAT” medium
and the two main pathway of the DNA synthesis
”salvage” pathway
reusage of nucleic acid degradation
products: purins, hypoxanthine, thymidine
HGPRT, TK
(hypoxanthine guanine phosphoribosyl transferase,
thymidine kinase)
DNS
nucleoside triphosphates
aminopterin
other enzymes
”basic” building blocks, simple sugars, amino
acids
”de novo” pathway
Only this pathway is
functioning in the SP2
myelome cells!
A slightly detailed picture:
”salvage” pathway
purins, e.g.
hypoxanthyne
thymidine
HGPRT
TK
dGTP
IMP
dTMP
DNS
dATP
phosphorybosyl
pyrophosphate,
glutamine
dTTP
dCDP
OMP
dCTP
aminopterin
”de novo” pathway
HAT  Hipoxantin, Aminopterin, Timidin
aspartic acid,
carbamoil phosphate
IMP – inozin monofoszfát
OMP – orotidin monofoszfát
HGPRT – hipoxantin-guanin foszforibozil transzferáz
TK – timidin kináz
(animated slide)
2nd main selection step:
Selecting the specific antibody producing cells
Antigen specific
antibody producing
hybridome cells
The antibodies in the cell
culture medium can react
with the antigen
Other antibody
producing or non
antibody producing
hybridome cells
No specific antibody in the
cell culture medium
Discarded
Further culturing,
Cell cloning
(animated slide)
Cell cloning
All hybridome cells
produce policlonal
(oligoclonal) antibodies.
Some non-specific
hybridome can also
exist among the
specific ones.
Only the antigen specific monoclonal antibody producing B cell
hybridome clones are needed
(checking e.g. ELISA method)
Difficulties, annoyances:
The transformed cells and the hybridomes with multiple
chromosome sets are genetically unstable  chromosome loss
They should be regularely tested for the antibody production.
- recloning
Establishing HGPRT and TK defficient
cell lines
Tumour cells can be genetically unstable
Spontaneous mutant cell lines can be effectively selected
Selecting HGPRT-, TK- (defficient) fusion partner cels
”Abortive” hipoxanthin and tymidine analogues can be used
for selecting HGPRT- and TK- cells
The HGPRT+ or TK+ cells incorporate these by the ”salvage” pathway
into the DNA and they die.
The HGPRT- and TK- cells can survive.
Comparison of mono- and polyclonal antibodies
Polyclonal
antibody
Low affinity
monoclonal Ab.
High affinity
monoclonal Ab.
Number of the
recognised epitops
many
one
one
Specificity (cross
reactivity)
polyspecific
usually polyspecific
monospecific
Affinity
low to high
(mixture!)
low
high
Non-specific
antibodies
many
0
0
yield (concentration)
High (serum)
Low (cell culture
supernatant)
Low (cell culture
supernatant)
Production cost
Low (depends)
high
high
Standardisation
No (or difficult)
simple
simple
quantity
limited
unlimited
unlimited
Practical usage
Depends from the
purpose
poor
excellent
The applications of the monoclonal antibodies in practice
DIAGNOSTICS
•Blood typing, tissue typing (anti-A, anti-B, anti-D antibodies)
•Analysis of complex antigenic mixtures (sera, liquor)
•Analysis of cell types and functions with cellular markers
•Immunohistochemistry
•Lymphoma typing by CD markers
THERAPEUTIC POSSIBILITIES (several hundred products exist)
•Cell separation
•CD34+ bone marrow stem cell separation
•Tumour therapy (e.g, targeted chemotherapy)
•anti CD20 antibodies against B cell Non-Hodgkin lymphoma
• Therapeutic inhibition of the cellular functions by blocking specific receptors or
the neutralisation of the ligands
•Tumour therapy
•Immunosuppression
•T cell suppression after tissue/organ transplantation
Passive immunisation
mouse monoclonal
antibodies
immunisation
humanized, mouse
monoclonal antibodies
(recombinant methods)
immunisation
PROTECTED
INDIVIDUAL
serum antibodies
(blood donation)
ENDANGERED
INDIVIDUAL
Human immunoglobulin
transgenic mouse
human monoclonal
antibodies
• Only the effector functions of the
immune response are activated
• Immediate effect
• Temporary protection
Immunoglobulin degradation!
Natural passive immunisation:
Human:
• Maternal IgG can be transported through the placenta by FcRn:
protection of the newborn in the first months
• Breast milk: IgA (by pIgR), some IgM, IgG : protection of the upper
gastrointestinal and respiratory tracts
Animals, household animals:
• Placental transfer? (not known)
• The colostrum of some animals contains high concentration of
maternal antibodies (IgG!) – can pass from the intestine into the
circulation of the newborn (various mechanisms: FcRn, ”leaky”
newborn intestinal epithelial cell layer)
Therapeutic usage of polyclonal antibodies
• Collected blood plasma for helping immunocompromised persons
• Antivenoms
antisera against the toxin of various venomous
animals: snakes, scorpions, spiders, venomous
sea creatures
o
Slow, gradual immunisation, and hyperimmunisation
of large animals (usually horses)
o
Separation of the gamma-globulin fractions from the
sera by biochemical methods: Decreases the
possibility of the patients’ immunisation against the
foreign plasma proteins. Repeated administration
may be possible.
Therapeutic antibodies
Monoclonal antibodies, as
medicaments?
The commonly available animal (mouse) antibodies can act
as antigens inside the human body (foreign proteins!!!)
How could it be coped with?
The „evolution” of the therapeutic monoclonal antibodies
mouse /rat
chimera
Human
Humanized
The glycosylation of the humanized antibodies are still similar to the producing cell type.
Real human monoclonals can be established by human hybridomes.
The most frequently used therapeutic
monoclonal antibody medicaments
- Tumourtherapy
Cell type specific targeting of the tumours by
selective chemotherapy
- Immunosuppression
Cell type specific immunosuppression, receptor
antagonists, ligand neutralisation
Monoclonal antibodies in tumour therapy
Immunosuppressive antibodies and
receptor-Fc fusion products
1. Anti-TNF-α antibodies
infliximab (Remicade): 1998, chimera
adalimumab (Humira): 2002, recombinant human
2. Etanercept (Enbrel) – dimer fusion protein,
TNF-α receptor + IgG Fc-part
IgG Fc-part:
increased half life and spreadability in the body by FcRn
Anti-TNF-α therapy:
• Rheumatoid arthritis
• Spondylitis ankylopoetica (SPA - M.
Bechterew)
• Psoriasis, arthritis psoriatica
• Crohn-disease, colitis ulcerosa
Antibody purification in affinity column
(a topic of the affinity chromatography)
column
polymer beads
column matrix
affinity purifed antibody
You can find antibodies in varios catalogs of biotech
companies which are purified this way.
chemically bound antigens
(animated slide)
antibody
containing
solution
binding
washing
elutionAntigen-specific
polyclonal antibodies
(animated slide)
It can be assembled in the opposite way:
Protein (antigen) purification with antibody column
column
polymer beads
antigen containing solution
binding
washing column matrix
elution
Purified antigens
chemically bound antigenspecific antibodies
The usage of superantigens for antibody purification
The bacterial protein A, or protein G superantigens are generally used
for the purification of monoclonal antibodies from the supernatant of the
hybridome cell cultures.
Protein A, Protein G  can bind to the Fc part of the antibodies
Protein L  can bind the kappa light chain part of the antibodies
Immunoglobulin binding specificity of protein A, G, and L
Species
Human
Rabbit
Cow
Cat
Horse
Goat
Guinea-pig
Sheep
Dog
Pig
Rat
Mouse
Chicken
Monkey(rhesus)
Hamster
Koala
Llama
IgG
IgG1
IgG2
IgG3
IgG4
IgA
IgA1
IgA2
IgD
IgE
IgM
IgG
IgG
IgG1
IgG2
IgG
IgG
IgG
IgG1
IgG2
IgG1
IgG2
IgG
IgG1
IgG2
IgG
IgG1
IgG2a
IgG2b
IgG2c
IgG3
IgG
IgG1
IgG2a
IgG2b
IgG3
IgM
IgY
IgG
Protein A
Protein G
Protein L
+++
++++
++++
++++
+
+
+
++
+
+++
+
+
+++
+++
++
+
+
+++
++
++
+
+
+++
++
+++
+
++
+
++
+
++++
+++
++
++++
+
-
+++
++++
++++
+++
++++
+++
+++
+++
+++
+
++++
++
+++
+++
+
+
++
++
+++
+
++
++
+
++++
++
++
++
++
++++
++++
+++
+++
++++
++
+
+
+++
++++
++++
+++
++++
+++
+++
+++
+++
+++
+++
+
?
?
?
?
?
+++
+++
+++
+++
+++
+++
?
+++
+++
+++
+++
+++
+++
?
+++
?
?
Source: Sino Biological Inc.: http://www.sinobiological.com/Recombinant-Protein-A-g-332.html?gclid=CK-QhN2vpMsCFYcp0wod9wUClQ
Immunprecipitation (IP)
Do not mix it with the precipitation
reaction after the formation of
large Ab-Ag immuncomplexes!
(see next lecture)
Useful in the identification of
protein-protein interactions:
The catched antigen will pull out
the interacting proteins also 
which can be identified later in
the WB as extra lanes.
Intracellular signalling cascades
can be examined with it. The
interacting signalling proteins in
the cascade can be revealed.
Practice
Polyclonal antibody response
Ag
Immunserum
B cell set
Activated B cells
Policlonal
antibodies
Antibody producing
plasma cells
Ag
Antigen-specific
polyclonal antibodies
(the products of different B cell clones!)
Immunsorbent column
Column
sepharose beads
BSA
(animated slide)
Applying mouse serum
Incubation
(BSA specific)
(animated slide)
Removing the non-bound serum components
(Washing step: indifferent serum proteins, non BSA-specific antibodies)
PBS
The color of the
Coomassie stain is not
switching to blue without
proteins
Coomassie
Brillant Blue
G250 protein
stain
(animated slide)
Elution of the BSA-specific antibodies
4-5 ml glycin-HCl
buffer
You can check the protein (BSA-specific
antibody) content of the eluates with
Coomassie protein stain: ocassional 1 dropp
eluate in a tube with Coomassie stain
The 9-10 dropps are collected
into TRIS-buffer containing
tubes (for neutralising the pH)
Recloning of the cells with limited dilution (fast-cloning):
1. Counting the hybridome cells
2. Distribute 100 l cell culture media into each well of a 96 well cell culture plate.
3. Put 1000-5000 cell in additional 100 l medium into thefirst (A1) well.
4. Serial dilution with 100 l (1:1) down the column.
5. Fill up the wells of the first column to 200 l.
6. Serial dilution of the first column to the right with 100 l (1:1) (multichannel pipette)
You can get single cell wells in the down-right side of the cell culture plate with high probability
Halving
dilutions
Halving
dilutions
A
B
C
D
E
F
G
H
1
2
3
4
5
6
7
8
9 10 11 12
Bürker chamber (3x3 or 4x4 grid)
The cumulative area of 25 small rectangle (an example is
labeled with red) or the area within the triple lines (see the red
area on the right side picture): 1 mm2
The distance between the coverslip and the chamber surface:
0.1 mm
The volume: 0.1 mm3 = 10-4 cm3 (ml)
Your counted cells in the 1mm2 area
× 104 × additional dilutions
= final cell number/ml
0.2×0.2=0.04mm2
1 mm
0.2 mm