C8116
Antibodies as immunochemical tools
Spring semester 2025
Hans Gorris
Department of Biochemistry
March 4th, 2025
2
Antigenic determinants: hapten
Carrier
Hapten
Other examples:
toxins, pharmaceuticals,
hormones…
• Immunization generates antibodies only against large molecules, e.g. proteins
• Antibodies against small molecules (haptens) must be produced by coupling
(typically derivatized) small molecule onto the surface a large carrier protein.
Definition of hapten:
A low-molecular weight
molecule which contains
an antigenic determinant
but which is not itself
antigenic unless bound
to an antigenic carrier
3
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
4
Generation of monoclonal antibodies
=> first described by Köhler/Milstein (1975)
(PEG or electrofusion)
(myeloma cells)
5
Generation of monoclonal antibodies
limiting dilution
(HAT)
6
Antibodies as immunochemical reagents
=> Antibodies are used
as bioanalytical reagents
to specifically detect and
quantify other molecules Epitope
paratope
7
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
8
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.
9
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
10
From heavy chain antibodies to nanobodies
heavy-chain antibody IgG antibody
VHH: Single variable
domain on a heavy
chain (=> nanobody)
11
Nanobodies: Detection of hidden epitopes
12
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
a Chromobodies.
b Detection of the nuclear lamina with lamin chromobody in living cells. Confocal images of
HeLa cells coexpressing lamin chromobody (green) and red fluorescent histone H2B as a
mitosis marker. Scale bar: 10 µm
13
Recombinant nanobodies
Anal. Bioanal. Chem. (2010) 397: 3203–3208
14
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)
15
Display of individual
protein variants on
the virus surface
Construction of phage displayed protein libraries
16
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
17
Single-domain antibody (nanobody)
Problem here:
the genetic information
for Vh and VL need
to be fused to get
functional paratops
18
Production of recombinant antibodies
Expression system
(Gene of Ab fragment known)
Phage display
(Gene of Ab fragment unknown)
19
Alternatives for antibodies
20
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
21
22
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
23
24
Molecularly imprinted polymer (MIP)
“Plastic antibodies“
25
Immunoassays
Literature for in-depth reading
26
A rough categorization of immunoassays
27
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
28
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
29
antibody
(capture)
antibody
(labelled)
analyte (=> antigen)
label
("sandwich" immunoassay)
Non-competitive immunoassay
solid phase
(enables easy
separation)
30
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
31
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
32
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
33
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
34
labeled antibody is bound;
excess is washed away
Non-competitive immunoassay
antibody
(capture)
antibody
(labelled)
solid phase
35
signal of the label is measured
Non-competitive immunoassay
antibody
(capture)
antibody
(labelled)
solid phase
36
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)
37
38
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.
39
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.
very
common
Enzyme Properties
peroxidase
galactosidase
rarely found in biosamples, high activity
phosphatase
glucose oxidase
rarely found in biosamples, moderate activity
catalase high activity but often present in samples
protease low activity
less
suitable
=> Effect: strong signal amplification
(one enzyme label generates 100 - 1000 chromophores / fluorophores per second!)
Enzyme-linked immunosorbant assay (ELISA)
40
(a) a chromogenic substrate (3,3‘,5,5‘-Tetramethyl-benzidine (TMB)):
Stops the enzyme reaction:
Endpoint measurement
(b) a fluorogenic substrate:
- Coloration depends on the amount of
enzyme-labeled secondary Ab;
- microtiter plate reader; absorbance
at 450 nm expressed as Optical
Density (OD)
blue yellowcolorless 1 M H2SO4HRP
Enzyme-mediated signal generation
41
Alternative non-competitive ELISA formats
42
one-step assay
Immunometric assay
=> analyte is detected directly,
i.e. signal from immune complexes containing analyte
43
When capture antibody
becomes saturated,
free analyte in solution
binds to the detection
antibody and prevents
it from binding to the
antigen on the solid
phase
Immunometric assay
one-step assay
44
Immunometric assay
two-step assay
=> avoids high
dose hook effect
45
4-parameter logistic function Variables: "optical density" (OD
= absorbance) and [An]
Fitted parameters:
• ODmax (signal at saturation)
• bg (background signal)
• C50 (midrange concentration)
• s (slope)
An-Ab binding:
[An] + [Ab] [AnAb]
k1
----->
<-----
k2
ELISA: data analysis
𝐾 =
[𝐴𝑛𝐴𝑏]
𝐴𝑛 [𝐴𝑏]
𝑂𝐷450 =
𝑂𝐷 𝑚𝑎𝑥 − 𝑏𝑔
1 +
𝐴𝑛
𝐶50
𝑠
Surface-bound
immune complex
reflects K
46
Bound analyte
[labeled detection Ab]
High affinity antibody
Low affinity antibody
Detection limit of non-competitive assay
Background
signal
95% of maximal binding
with high S/N ratio
95% of maximal binding
with low S/N ratio
47
Limit of detection (LoD) vs. limit of quantification (LoQ)
LoD LoQ
The smallest concentration of an analyte in
a test sample that we can easily distinguish
from zero
The smallest concentration of an analyte in
a test sample that we can determine with
acceptable repeatability and accuracy
Immunometric assay
dose-response curve
48
Optimization of immunoassays
[Analyte] (10-n M) Signal(OD)
Signal(OD)
optimal range for measurements
(specific window)
specific non-specific
non-specific
specific
[Analyte] (10-n M)
49
Non-optimized assay Optimized assay