C8116 Immunochemical techniques
Immunoassays
Spring term 2024
Hans Gorris
Department of Biochemistry
March 26th, 2024
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2
Antibodies as immunochemical reagents
=> Antibodies are used
as bioanalytical reagents
to specifically detect and
quantify other molecules Epitope
paratope
From heavy chain antibodies to nanobodies
our own most
common antibody
heavy chain antibodies
(velbloud, dromedár, lama) (žralok)
Front. Immunol., 2017 https://doi.org/10.3389/fimmu.2017.00977
3
Phage display
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Aptamers
KA: 109
RNA or DNA aptamer
Complementatary
base pairing
Binding through:
(1) 3-dimensional, shape-dependent interactions
(2) hydrophobic interactions, base-stacking, intercalation
5
Molecularly imprinted polymer (MIP)
“Plastic antibodies“
MIPs are generated by:
polymerization of monomers in presence of the template analyte
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7
Immunoassays
A rough categorization of immunoassays
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antibody
(labelled)
analyte (=> antigen)
label
Sandwich immunoassay
9
antibody
(capture)
excess of binding sites
solid-phase
(enables easy
separation)
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)
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(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
11
Alternative non-competitive ELISA formats
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one-step assay
Immunometric assay
=> analyte is detected directly,
i.e. signal from immune complexes containing analyte
13
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Immunometric assay
one-step assay
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Immunometric assay
two-step assay
893&6-+.1 "+7"
.-1# "--:
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15
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]
𝑂𝐷!"# =
𝑂𝐷$%& − 𝑏𝑔
1 +
𝐴𝑛
𝐶"#
'
Surface-bound
immune complex
reflects K
16
Bound analyte
;5&,#5#.3.#(#%(+-$3<,=
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
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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
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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)
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Non-optimized assay Optimized assay
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Labeling strategies
Fluorescence/luminescence
=> high-specific activity
Signal amplification Background reduction
- brighter fluorescence
improved quantum yield / quantum dots
- electrochemistry
amperometry, voltametry, impedimetry
- multiple labeling
attaching several reporter molecules /
dye-doped nanoparticles / liposomes
- (electro-)chemiluminescence
luminol / ruthenium-bipyridyl-complex
- signal cascades
subsequent amplification steps
- time-resolved fluorescence
lanthanide complexes
- enzyme amplification
horseradish peroxidase
- anti-Stokes photoluminescence
photon-upconversion, UCNPs
S ↑ B ↓
S/B
Here we only talk about the optical signal and background
=> even the best S/B is useless if the label binds to the surface
(non-specific binding)
Various detection modes can
be combined with each other
Signal cascades
Advantages:
- only one type of secondary antibody is needed for many types of primary antibodies
- higher sensitivity => polyclonal secondary antibody can bind
to different sites of the primary antibody
S (signal) examples:
- radionuclide (radioimmunoassay)
- fluorophore (fluorescence microscopy)
- enzymatic (ELISA)
S
direct indirect
detection
primary
antibody
=> mouse IgG
secondary
antibody
=> anti-mouse IgG
analyte (antigen)
biotin
streptavidin
signal
amplification
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S
S
Capture
antibody
PCR
Fluorescent
nucleotides
DNA
Secondary
antibody
Analyte
But:
Even the best immunosassay is
less dependent on the highest
possible signal than on the ratio
of signal-to-noise (SNR).
Signal Amplification by PCR
Immuno-PCR
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23
Chemiluminescent labeling
Weeks, I. (1983) Clin. Chem. 29, 1474-1479
=> no enzyme required, but only one photon per molecule
(low specific-acitivity label)
acridinium ester
24
Chemiluminescent labeling
Emission of 425-nm light (violet)
luminol
=> HRP oxidizes luminol:
one photon per catalytic turnover event
25
Electro-chemiluminescent labeling systems
=> Light emission mediated by a redox reaction
Ru(bpy)3
2+: Ruthenium bipyridyl complex
TPrA: Tripropylamine
Emission of 620nm
light (orange)
=> no enzyme required
=> the label can cycle between an oxidized and reduced state
to generate many photons per molecule
26
(Electro-)chemoluminescent labels
Advantages: no need for excitation light
=> Simpler and more compact instrumentation
=> No autofluorescence or light scattering: background-free detection
Disadvantages:
Each (electro-)chemical or enzymatic turnover event
only results in the emission of a single photon
=> weaker overall signal / not the highest activity
(as compared to fluorescence)
27
Time-resolved lanthanide fluorescence
=> Guest lecture on April 23rd
Photon-upconversion nanoparticles
(UCNPs)
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wavelength (nm)
29
van de Rijke et al. (2001) Nature Biotechnology 19, 273–276
UCNPs: anti-Stokes emission
Sequential absorption of 2 or more photons via long-lived transition states
=> More time for absorbing a further photon 30
Sequential absorption of two or more photons
NaYF4:Yb,Er NaYF4:Yb,Tm
Near-infrared excitation (980 nm)
Luminescence of UCNPs
depends on lanthanide
dopant composition
Upconversion luminescence of UCNPs
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32
Emission depends on lanthanide composition
In time-resolved
measurements:
different lifetimes
Here:
different emission
signatures
Anti-Stokes shift
Emission
green
red
Photon-upconversion is ca. 1,000,000 x
more efficient than 2-photon excitation
=> excitation by using a continuous 980-nm laser source
TEM of UCNPs
NaYF4:Yb,Er
Hexagonal
crystal structure
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UCNPs as background-free optical labels
Emission
green
red
NIR-excitation
Optical window
No autofluorescence
Very low light scattering
... and completely photostable
Background-free imaging
TEM of UCNPs
NaYF4:Yb,Er
Hexagonal
crystal structure
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UCNPs as background-free optical labels
Features Unlike Enables
Excitation by NIR light
(980 nm)
Organic fluorophores
/ QD
1) Background-free diagnostic assays
2) Deep tissue / small animal imaging
Large anti-Stokes shifts Org. fluorophores Excellent separation of detection channels
Narrow and multiple
emission bands of
UV, visible or NIR light
Org. fluorophores Multiplexing / ratiometric measurements
No photobleaching Org. fluorophores Long-time imaging
Paramagnetic
(co-dopant: Gd3+)
Org. fluorophores Hybrid nanoparticles:
Magnetic resonance imaging (MRI)
Low toxicity QD / radionuclides Cellular imaging / easier handling
Advantages of UCNPs
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Microsocope Microtiter plate reader
Instruments for the detection of UCNPs
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Hlavacek, A. (2022) Nat. Prot. doi: 10.1038/s41596-021-00670-7
Surface functionalization of UCNPs
i
k
Si O
O
O
Si
O
O OH
Si
O
O O
Si
O
O
O OH
–
Si
O
O
Si
O
O
Si
O
O
Si
O
O
O
NH
Si
O
O
O
O
CH3
CH3
CH3
CH3
Si O
O
–
OH
OH
OH
Na
+
N3
H2N
a
c
e
g h
j
f
d
NaREF4
Si O
O
–
O
O
Si
O
O OH
Si
O
O O
Si
O
O
O OH
–
Si
O
O
Si
O
O
Si
O
O
Si
O
O
O
CH3
O
O
NaREF4
NaREF4
NaREF4
b
(
)
~110
O
NH
O
PP
OH
O
O
–
O
O
–
O
–
O
–
(
)
~110
O
NH
O
PP
OH
O
O
–
O
O
–
O O
~110
N
N
N
O
NH
O
PP
OH
O
O
–
O
O
–
O O
(
)
NaREF4
NaREF4
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Microtiter plate
Mickert MJ (2019) Anal. Chem. 91, 9435
Upconversion-linked immunosorbent assay (ULISA)
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Gold nanoparticles
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40
Introduction to gold nanoparticles (colloidal gold)
- known since ancient times (glass staining)
- modern synthetic approaches: size control in the range of 2 to 100 nm
- synthesis: reduction of HAuCl4 in aqueous solution e.g. by citrate
- simple surface modification e.g. via self-assembled monolayer (SAM): thiols
- properties:
1. chemically stable
2. high electron density
3. collective oscillations of valence electrons in metal grid
in resonance with frequency of visible light
absorption by localised
surface plasmon resonance
size-dependent
(gold) nanoparticle-labeled
detection antibody
nitrocellulose
membrane
test
line
control
line
immobilized
capture antibody
test line
immobilized
anti-antibody
antibody
application pad flow control line
sample / analyte
Flow direction
• Separation-based assay using capillary flow in nitrocellulose membrane
• qualitative result: yes/no answer
• pregnancy test measures hCG (human chorionic gonadotropin)
Lateral flow assay
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test
line
control
line
(1) Analyte is bound to labeled antibody
(2) Analyte-labeled antibody complex and non-bound labeled antibody move with flow
test
line
control
line
test
line
control
line
(3) Analyte-labeled antibody complex is bound to immobilized capture antibody;
labeled antibody is bound to immobilized anti-antibody antibody
Lateral flow assay
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sample
liquid flow
1st line (test result)
(analyte + labeled antibody)
-> if not visible, HCG not high
-> not pregnant
2nd line (control)
-> proof that flow and labeled
antibodies have worked
Lateral flow assay
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Excursion: Biosensors
Drápatka vodní
(Xenopus laevis)
Injection of sample urine (containing hCG) to the dorsal lymph sac of femal frog
=> Frog starts ovulation within 12 hours
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=> an example of a “biosensor“
Scheme of a biosensor
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Bacteria
Diagnostic
markers
Viruses
Toxins
Analyte
Signal
transducer
Bioanalytical
information
Biorecognition
element
e.g. antibody
=> What is the difference between
an immunoassay and a biosensor?
A rough categorization of immunoassays
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Note: The sandwich ELISA is not applicable to small molecules such as
steroid hormones (e.g. progesterone), because they do not possess two
epitopes for binding both the capture Ab and the detection Ab.
Competitive immunoassay
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solid phase
antibody
limited amount of antibodies
antibody against analyte either recognizing
• single epitope = monoclonal ab, or
• multiple epitopes = polyclonal ab
Competitive immunoassay
48
analyte and labeled analogue are
added; incubation for binding
Competitive immunoassay
solid phase
antibody
limited amount of antibodies
analyte labeled
analyte-
analogue
(tracer)
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competition in binding to a limited number of antibodies
non-bound analyte and
analogue are washed away
Competitive immunoassay
solid phase
antibody
analyte
Labeled
analyte-
analogue
(tracer)
50
signal of the label is measured
Competitive immunoassay
solid phase
antibody
limited amount of antibodies
51
• First kind of immunoassay: 1950s (Rosalyn Yalow, Nobel price in 1977)
and still in use (very sensitive and background-free)
• In addition to the analyte, a second antigen that carries a radioactive (“hot”)
label is needed (concentration must be known) => tracer
• Radionuclides: typically 125I, 3H => safety precautions are needed
• Mainly used for small molecule analytes such as hormones in a competitive
immunoassay (originally developed for insulin), other example: renin, a
marker for hypertension (concentration in serum: 10-12 M)
Radioimmunoassay (RIA)
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Radioimmunoassay (RIA)
Boundradiolabeledantigen(%)
Labeled (“hot“) antigen
Unlabeled (“cold“) antigen
Cold antigen (pg)
Unknown antigen concentration can be determined
from standard curve
Solid phase
Antibody specific for a
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• On the first sight:
radionuclides are perfect labels for immunoassays => background-free
(there is no intrinsic radioactivity in sample, test tube of instrument)
54
Gorris & Soukka, Anal. Chem. 2022, 94, 6073
Radioimmunoassays (RIA)
• It is obvious: radioactive labels (e.g. 125I, 3H) require special safety precautions
• But also:
Each decay event of a radionuclide is detectable only once
125I (gamma rays): t1/2 = 60 days / 20 - 48% of radiation is detected
è if there is one radiolabel per detection antibody molecule, more than 2500
labeled Ab molecules are needed to detect one decay event / hour
è “low-activity“ labels need long signal acquisition times
• Using nuclides of shorter half-lives (providing more decay events / second)
is not an option because
=> their shelf life is reduced accordingly
=> a higher activity leads to radiodamage of biomolecules
Competitive immunoassay
solid phase separation
=> analyte is measured indirectly
i.e. signal from those binding sites where the analyte is absent 55
LOD
(Limit of
detection)
3xSD
=> typically not as sensitive (i.e. higher LOD) than immunometric immunoassay
Competitive immunoassay
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100
)(
)(
(%)
50
50
x
IC
IC
CR
reactantcross
analytemain
=
Cross reactivities (%) for structurally related compounds are determined in
comparison to the main analyte at test mid points (IC50) and are expressed in %:
High cross reactivity: CR (%) > 10
Low cross reactivity: 1 < CR (%) < 10
No cross reactivity: CR (%) < 1
!"#$%&'($)*+',-#.+ /0012'/343/15361
Cross reactivities
Hlavaćěk (2016) Anal. Chem. 88, 6011-6017
Competitive ULISA I
58
Peltomaa, R. (2020) Biosens. Bioelectron. 170, 112683
Competitive ULISA II
59
Peltomaa, R. (2020) Biosens. Bioelectron. 170, 112683
Competitive ULISA II
60
vs.
Competitive immunoassay: alternative formats
61
Labeled analyte serves as tracer Labeled antibody serves as tracer
Determination of saliva immunoglobulin IgA
(this test is commercially available)
Steps:
1) prepare an antibody-enzyme conjugate
2) add to analyte solution in excess
3) make a microplate with immobilized IgA
4) add solution
5) let react; wash; add substrate
6) measure color
Indirect competitive immunoassay
62