C8116
Antibodies as immunochemical tools
Spring semester 2024
Hans Gorris
Department of Biochemistry
March 18th, 2024
2
Summary of interplay between TH and B cells
3
Antibodies as immunochemical tools
Immunology
The “tools“:
antibodies Immunoassay
4
Polyclonal vs. monoclonal antibodies
polyclonal monoclonal
Antibodies that are collected
from sera of exposed animal
recognize
multiple antigenic sites
of injected substance
Individual B cell hybridoma is cloned and
cultured.
Secreted antibodies are collected from
culture media
recognize
ONE antigenic site
of injected substance
Generation of monoclonal antibodies
5
Immunogen: Molecule that is capable of eliciting an immune response by
the immune system of an organism.
Antigen: Molecule that is able to bind to the
product of that immune response: the
antibody.
Antibodies, or immunoglobulins (Igs), are g-globulin proteins folded into well
defined three-dimensional structures synthesized by living organisms, e.g.
mice, rabbits or goats, or by living cells, in response to the presence of a
foreign substance known as the antigen.
Hapten: Small molecules (< 5000 Dalton) that need to be conjugated to a
carrier protein (e.g. bovine serum albumin (BSA), keyhole limpet
hemocyanin (KLH) or ovalbumin) to elicit the immune response.
Epitope: An epitope is a specific location on
the surface of an antigen that has a
particular molecular structure and that is
recognized by a particular antibody or a set
of specific antibodies that the epitope elicits
during the immune response.
Antibodies: Definitions
Antibodies: Definitions
!""#$%&'$( )*' )+,)-( )$./&'$( 0#. $%. )++ )$./&'$( )*' /""#$%&'$(
HAPTEN SPACER
IMMUNOGENIMMUNOGEN
CARRIER
HAPTEN SPACER
IMMUNOGENIMMUNOGEN
CARRIER
SPACER BINDING EFFECTSPACER BINDING EFFECT
HAPTEN SPACER
CARRIER
Aff1Aff2
AntibodyAntibody
binding sitebinding site
SPACER BINDING EFFECTSPACER BINDING EFFECT
HAPTEN SPACER
CARRIER
Aff1Aff2
AntibodyAntibody
binding sitebinding site
/ Protein (e.g. KLH)
/ Protein (e.g. KLH)
8
Antibodies as immunochemical reagents
=> Antibodies are used
as bioanalytical reagents
to specifically detect and
quantify other molecules Epitope
paratope
9
Continuous vs. discontinuous eptiopes
peptide chain
epitope
antibody binding/recognition site
Continuous epitope:
short peptide or denatured protein
structure, epitope consist of
sequential amino acids
Discontinuous epitope:
present only in 3-dimensional
protein structure, epitope comprises
non-sequential amino acids
peptide chain
epitope
10
Excursion: Epitope mapping
How do we know to what epitope an antibody binds?
11
Epitope mapping
=> But only
continuous epitopes
12
Overlapping eptiopes
Even small analytes can have multiple epitopes, but antibody binding to one
epitope blocks another epitope, i.e. these epitopes are overlapping
short peptide
epitope-2
epitope-1
antibody-1
antibody-2
13
Non-overlapping eptiopes
antibody-1
epitope-1
epitope-2
paratope-2paratope-1
antibody-2
a folded protein
“the analyte”
14
Monoclonal antibody reagent
all antibodies are from the same B cell clone
=> reagent consist of identical antibodies,
and all recognize and are specific
for only one identical epitope
identical
paratopes
epitope-1
epitope-2
.. will bind only to one specific
epitope in the analyte - unless
there are multiple identical
epitopes in the same analyte
15
Polyclonal antibody reagent
Mix of different B cell clones
=> reagent consist of antibodies that have
different paratopes and recognize different
epitopes, but exact composition is not known
different
paratopes
epitope-1
epitope-2
… will bind simultaneously to
one or several non-overlapping
epitopes in the analyte
epitope-3
16
Antibody affinity
17
Affinity of an antibody
Tight fit /
high affinity
Less interaction
/ lower affinity
Little interaction /
very low affinity
good epitope specificity decreasing epitope specificity
18
Antibody-antigen binding reaction
Ab (antibody) Ag (antigen/analyte)
+ ®
¬
AgAb
Ag + Ab AgAb
ka
kd
19
Surface plasmon resonance (SPR)
antibody
attached to
gold film
solution of antigen molecules
20
Determining the affinity of antibodies by SPR
(1) Binding of antigen to surface immobilized antibodies
increases the refractive index of the surface layer.
(2) The resulting change of the resonance angel for
plamson induction can be measured by a photodetector.
solution
of antigen
molecules
buffer
solution
antigen
added
buffer
wash
21
Affinity of an antibody
reaction velocities at equilibrium:
ka: association rate constant (on rate)
kd: dissociation rate constant (off rate)
K: affinity constant
𝑘! 𝐴𝑔 𝐴𝑏 = 𝑘" 𝐴𝑔𝐴𝑏Ag + Ab AgAb
ka
kd
𝐾 =
𝑘!
𝑘"
=
𝐴𝑔𝐴𝑏
𝐴𝑔 𝐴𝑏
Bound antibody
and antigen
free antibodyfree antigen
22
Affinity of an antibody
Sensorgrams for 3 moncolonal antibodies against HIV p24 surface Ag
poorly suited for immunoassay
due to dissociation
problems, e.g. during
wash or during 2nd step
if used as capture antibody
extremely stable binding:
off-rate is very slow
23
Affinity of an antibody
approximate calculation of concentrations in equilibrium:
if [Ag]tot << [Ab]tot, only a very small antibody fraction is present in the complex [AgAb]
=> [Ab] » [Ab]tot
[Ag]tot = [Ag] + [AgAb]
[Ab]tot = [Ab] + [AgAb]
free (unbound) concentrations
𝐾 =
𝑘!
𝑘"
=
𝐴𝑔𝐴𝑏
𝐴𝑔 𝐴𝑏
Bound antibody
and antigen
free antibodyfree antigen
𝐴𝑔𝐴𝑏 =
𝐴𝑏 #$# 𝐴𝑔 #$# 𝐾
𝐴𝑏 #$# 𝐾 + 1
24
Affinity of an antibody
[Ab]tot = 1 * 10-9 M
[Ag]tot = 1 * 10-12 M (i.e. much smaller)
K = 1 * 109 M-1
by calculating we get [AgAb] = 0.5 * 10-12 M (i.e. 50%)
"rule of thumb”: when [Ab]tot = 1/K then [AgAb] = 50% [Ag]tot
[Ab]tot = 10/K then [AgAb] = 90% [Ag]tot
[Ab]tot = 0.1/K then [AgAb] = 10% [Ag]tot
Calculating the equilibrium concentration
𝐴𝑔𝐴𝑏 =
𝐴𝑏 #$# 𝐴𝑔 #$# 𝐾
𝐴𝑏 #$# 𝐾 + 1
25
Affinity of an antibody
affinity increases
26
Affinity of an antibody: Scatchard plot
K ([Ab]tot - [Ab-Ag]) = =
[Ab-Ag] bound Ag
([Ag]tot - [Ab-Ag]) free Ag
[Ab-Ag]
([Ag]tot - [Ab-Ag])
[Ab-Ag]
slope = -K
[Ab]tot
bound Ag
bound Ag /
free Ag
linearization:
27
Affinity of an antibody: Scatchard plot
B = [Ag-Ab] F = [Ag] = [Ag]tot – [Ag-Ab]
K ([Ab]tot – B) = B/F => B/F = – K B + K [Ab]tot
constant
linear equation:
y = -kx + a
=> Typcially replaced by non-linear
fitting using computer programs
slope = -K
[Ab]tot
(when B = [Ab]tot, then B/F is 0)
B
B/F
28
Antibody engineering
29
Excursion: Antibody enginering for therapy
Natural antibodies
(raised in mice) are
potentially immunogenic
=> Potential side effects
30
Therapeutic antibodies (market value)
31
Humanized antibodies
No. Drug Indication (1st US FDA Approval Year) Company 2018 Revenue (USD, billion)
1Adalimumab Rheumatoid arthritis (2002) AbbVie $19.9 bn
(Humira) Psoriatic arthritis (2005)
Ankylosing spondylitis (2006)
Juvenile Idiopathic Arthitis (2008)
Psoriasis (2008)
Crohn’s disease (2010)
Ulcerative colitis (2012)
Hidradenitis suppurativa (2015)
Uveitis (2018)
2Nivolumab Melanoma (2015) Bristol-Myers Squibb $7.6 bn
(Opdivo) Non-small cell lung cancer (2015)
Renal cell carcinoma (2015)
Head and neck squamous cell (2016)
3Pembrolizumab Melanoma (2014) Merck & Co $7.2 bn
(Keytruda) Head and neck cancer (2016)
Non-small cell lung caccer (2015)
Lymphoma (2018)
Cervical cancer (2018)
Microsatellite instability-high cancer (2018)
4Trastuzumab Breast cancer (1998) Roche $7.0 bn
(Herceptin) Gastric cancer (2010)
5Bevacizumab Colorectal cancer (2004) Roche $6.8 bn
(Avastin) Non-small cell lung caccer (2006)
Breast ERB2 negative cancer (2008)
Renal cell carcinoma (2009)
Glioblastoma (2011)
6Rituximab, Non-Hodgkin’s lymphoma (1997) Roche $6.8 bn
32
Recombinant antibody fragments
VH
VL
CH1
CL
N
C
N
C C
N
S S
scFv
Fab
Mab
Disadvantage of antibodies as
a reagent: relatively large size
(Single-chain
variable fragment)
Flexible
peptide linkerVL VH
VL VH
CL CH1
Paratope
Artificial smaller constructs
Natural IgG antibody
=> same paratope,
but much smaller
33
Recombinant antibody fragments
Immortalization of hybridomas through cloning
or
generation of new antibodies without immunization
• Greater speed of production (E. coli batch fermentation)
• New specificities especially for poor immunogens
• Possibility to fine-tune antibody specificity and affinity
• Possibility to tailor make the antibody to perform special tasks
• tags, handles (for conjugation, immobilization)
• fusing to other protein (e.g. enzymes)
Likely to be increasingly used in miniaturised systems to enable full control
of antibody performance.
34
Heavy chain antibodies
our own most
common antibody
heavy chain antibodies
(velbloud, dromedár, lama) (žralok)
Front. Immunol., 2017 https://doi.org/10.3389/fimmu.2017.00977
35
From heavy chain antibodies to nanobodies
heavy-chain antibody IgG antibody
VHH: Single variable
domain on a heavy
chain (=> nanobody)
36
Nanobodies: Detection of hidden epitopes
37
Advantages of nanobodies
- Mass: ca. 15 kDa (IgG: 150 kDa), 2.5 nm diameter (IgG 15 nm)
- High solubility
- Rapid targeting and fast blood clearance
- Detection of “hidden“ epitopes
- Easy cloning: Recombinant engineering and protein expression in vitro in
bacterial production systems are much simpler
- Very stable and heat resistant (no cold storage required)
- Simple genetic structure allows easy re-engineering of nanobodies to
introduce new antigen-binding characteristics or attach labels
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8*9! /*33+ /%*:;$*++)07 3!&)0 /#$%&%'%(5 <7$**0= !0( $*( 132%$*+/*0. #)+.%0* 8>? !+ !
&).%+)+ &!$@*$, A/!3* '!$B CD E&
38
Recombinant nanobodies
Anal. Bioanal. Chem. (2010) 397: 3203–3208
39
Phage display using filamentous phage M13
• Infects / replicates in E. coli
• Protein coat:
major coat protein: pVIII
minor coat proteins: pIII, pVI, pVII, pIX
• The phage can be engineered to display foreign
peptides or proteins as a fusion with one of the coat
proteins, most commonly pIII.
• The genomic DNA encoding for the coat proteins is
enclosed within the protein coat.
=> Each protein remains connected to its encoding DNA
George Smith / Greg Winter:
Nobel prize in chemistry 2018
virus DNA
(circular and
single-stranded)
40
Display of individual
protein variants on
the virus surface
Construction of phage displayed protein libraries
41
Selection cycle Identification of high affinity
protein ligands
Option to introduce
random mutations
(e.g. error-prone PCR)
=> up to 1010
phage clones
Protein engineering by in vitro evolution
42
Single-domain antibody (nanobody)
43
Production of recombinant antibodies
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-.%)%/(0 12/0$34,%)+5)(6)7
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44
Alternatives for antibodies
45
Aptamers
KA: 109
RNA or DNA aptamer
Complementatary
base pairing
Binding through:
(1) 3-dimensional, shape-dependent interactions
(2) hydrophobic interactions, base-stacking, intercalation
SELEX*
*systematic evolution
of ligands by exponential
enrichment
=> in vitro selection
46
47
SELEX*
Step 1: Bind oligonucleotide
library and discard non-binder
Step 2: Elute oligonucleotides
that bind desired targets
Step 3: Perform PCR
to amplify eluted binders
Step 4: Repeat steps 1
through 3 using enriched
oligonucleotide pool
Aptamers: Assay designs
48
49
Molecularly imprinted polymer (MIP)
“Plastic antibodies“
Immunoassays
50
Literature for in-depth reading
51
“point-of-care testing“
History of immunoassays
52
“point-of-care testing“
History of immunoassays
53
A rough categorization of immunoassays
54
Solid phase matrix
Performance-related issues:
1) low background in detection system
2) immobilization qualities:
• high capacity
• suitable and easy coupling chemistries
• large surface
• maintained reactivity of capture protein
• no leakage
3) easy handling
4) inert in binding the labelled antibody/analyte => low background
5) effectively washed => low background
6) antibody excess through high density - surface measurement
7) antibody excess through large surface - integrating measurement
in heterogeneous non-competitive sandwich immunoassays
55
Size Examples Advantages Disadvantages
Small particle /
“beads”
(< 20 µm)
Latex
Microcrystalline cellulose
Fine porous glass
Magnetic beads
Liposomes
StarburstTM dendrimers
Dispensing as for liquids
Agitation not required
High antibody binding
capacity
Centrifugation required (unless
used with a membrane capture)
Long magnetic precipitation
Medium particle
(< 1 mm)
Sepharose beads
Sephacryl beads
Sephadex beads
Centrifugation not required
Short magnetic separation
Agitation required
Slower binding kinetics than above
Moderate antibody binding capacity
Single particle
(> 1 mm)
Polystyrene
Nylon
Centrifugation not required
Agitation not required
Some variability in antibody
coupling
Lower antibody binding capacity
Difficulty in dispensing
Poor binding kinetics
Fibers Membranes
Glass fibers
Nylon
Silicon rubber
Centrifugation not required
Agitation not required
No dispensing of reagent
Simple to use
Medium antibody binding capacity
Can be fast binding kinetics
Solid surface Coated tubes
Dipsticks
Microtiter plates (MTP)
Centrifugation not required
Agitation rare
No dispensing of reagent
Simple to use
Variability in antibody coupling
Lowest antibody binding capacity
Slowest binding kinetics
Solid phase matrices
Most frequently used
solid phase matrices
56
("sandwich" immunoassay)
Non-competitive immunoassay
57
antibody
(capture)
antibody
(labelled)
analyte (=> antigen)
label
("sandwich" immunoassay)
Non-competitive immunoassay
solid phase
(enables easy
separation)
58
solid phase
antibody
(capture)
excess of binding sites
a capture antibody specific for a
single epitope of the analyte is
coated on a solid phase
(e.g. on a microtiter plate)
(=> monoclonal antibody preferred)
Non-competitive immunoassay
59
sample containing the analyte (at
least two non-overlapping epitopes)
is added; incubation for binding
Non-competitive immunoassay
antibody
(capture)
excess of binding sites
solid phase
analyte
60
analyte is bound; in two-step assay:
sample is washed away with excess
of analyte
Non-competitive immunoassay
antibody
(capture)
excess of binding sites
solid phase
61
excess of labeled antibody (that
recognizes second epitope of the
analyte) is added; incubation for
binding
antibody
(labelled)
Non-competitive immunoassay
antibody
(capture)
excess of binding sites
solid phase
62
labeled antibody is bound;
excess is washed away
Non-competitive immunoassay
antibody
(capture)
antibody
(labelled)
solid phase
63
signal of the label is measured
Non-competitive immunoassay
antibody
(capture)
antibody
(labelled)
solid phase
64
Non-covalent
absorption of
capture
antibody to
polystyrene
surface
(microtiter plate)
Block surface
with BSA or
detergents to
prevent nonspecific
binding
of other proteins
Add sample that
contains the antigen
(the analyte), e.g.
tumor markers, viruses,
or antibodies in serum.
1. Add enzyme-labeled
detection antibody
(e.g. horseradish
peroxidase); wash
2. Add chromogenic
reagent (e.g. TMB)
3. Add "stop solution"
(e.g. H2SO4)
Thorough washing steps required
Enzyme-linked immunosorbant assay (ELISA)
65
66
Blocking is essential to avoid non-spec. binding
Normal serum
Normal serum (1-5% w/v) carries antibodies that bind to reactive sites and prevent
non-specific binding of the secondary antibody. Serum is rich in albumin and other
proteins that readily bind to non-specific protein binding sites of the sample.
Protein solutions
Blocking buffers often contain proteins such as bovine serum albumin (BSA),
gelatin or nonfat dry milk (1-5% w/v). These inexpensive and readily available
proteins are present in large excess compared to the antibody, so they compete with
the latter for binding to nonspecific sites in the sample. Many labs developed
homemade blocking buffers. It is important that blocking buffers are free of
precipitates and other contaminants that can interfere with the detection.
Commercial buffers
Ready-made blocking buffers can contain highly purified single proteins or
proprietary protein-free compounds. Many options are available that perform better
than gelatin, casein or other proteins used alone, and they have improved shelf lives
compared to homemade preparations.
67
Blocking tips
• Monitor both background (negative control) and signal strength (positive control)
with various blocking reagents.
• Choose the blocking buffer that yields the highest signal-to-noise ratio.
• Ensure that there are no substances in the blocking buffer that interfere with a
particular assay. Non-fat dry milk, for example, contains biotin and is
inappropriate for use with any detection system that includes a biotin-binding
protein.
• For optimal assay conditions, use the same blocking buffer for diluting the
antibody that is used for the blocking step.