C8116 Immunochemical techniques
Immunoassays II
Spring term 2025
Hans Gorris
Department of Biochemistry
March 17th, 2025
1
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
2
Phage display
3
Aptamers
KA: 109
RNA or DNA aptamer
Complementatary
base pairing
Binding through:
(1) 3-dimensional, shape-dependent interactions
(2) hydrophobic interactions, base-stacking, intercalation
4
Molecularly imprinted polymer (MIP)
“Plastic antibodies“
MIPs are generated by:
polymerization of monomers in presence of the template analyte
5
antibody
(labelled)
analyte (=> antigen)
label
Sandwich immunoassay
6
antibody
(capture)
excess of binding sites
solid-phase
(enables easy
separation)
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)
7
Non-optimized assay Optimized assay
8
Labelling systems in heterogeneous
immunoassays
9
New label designs
=> for different reasons and objectives
• higher specific activity
• “smart” reporters, that can e.g. distinguish
between specific and non-specific binding
• simplified / rapid assay formats, e.g.
homogeneous assays (“mix and measure”)
• simplified / low-cost / handheld detection
instruments
lower limit of
detection (LOD)
point-of-care
detection (POC)
• On the first sight:
radionuclides are perfect labels for immunoassays => background-free
(there is no intrinsic radioactivity in sample, test tube of instrument)
10
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
➔ if there is one radiolabel per detection antibody molecule, more than 2500
labeled Ab molecules are needed to detect one decay event / hour
➔ “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
11
Limit of detection (LOD)
=> limited by the specific activity of the label
[Ag]tot
R
kSD<R0>
[Ag]tot, lod
response
limit of detection
log
log
12
High-specific activity labels
solid-phase
antibody
(capture)
antibody
(labelled)
analyte
Low-specific activity label
=> higher signal for
each recognized analyte
High-specific activity label
?
13
Limit of detection (LOD)
=> Improvement afforded by high-specific activity labels
[Ag]tot
R
kSD<R0>
[Ag]tot, lod
response
limit of detection
log
log High-specific acitivity
(e.g. luminescent labels)
Low-specific acitivity
(e.g. radiolabels)
14
Non-specific binding
non-specific binding of labeled component (and
its variation) defines the actual limit of detection
non-
specific
specific
=> assay background over instrument and material background
15
Limit of detection (LOD)
=> limited by non-specific binding
[Ag]tot
R
kSD<R0>
[Ag]tot, lod
response
limit of
detection
log
log
Low non-specific binding
High non-specific binding
(e.g. insufficient blocking)
16
Strategies for high-specific activity labeling
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):
- 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
17
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
18
19
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
20
Chemiluminescent labeling
Emission of 425-nm light (violet)
luminol
=> HRP oxidizes luminol:
one photon per catalytic turnover event
21
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
22
(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
23
Time-resolved lanthanide fluorescence
24
Time-resolved fluorescence
"Nanosecond" fluorescence => decay time a few ns
e.g. - organic fluorophores
- quantum dots (somewhat longer decay times than organic fluorophores
"Microsecond" fluorescence => decay time 1 µs to 1 ms
e.g. - luminescent lanthanide labels (Eu, Tb, Dy, Sm, Er, Yb, Nd)
- metalloporphyrins (Pd, Pt)
Phosphorescence => decay time 1 ms to 100 s
25
Time-resolved fluorometry on lanthanide labels
• efficient reduction of background fluorescence
• concentration-dependent quantitative signal response
=> highly sensitive and reliable label detection
Problem: lanthanide ions (Ln3+)
• have only small absorption cross sections (ε < 1 M−1cm−1) compared to
fluorophores (ε up to 100 000 M−1cm−1)
• are strongly affected by water quenching (low quantum yield)
Solution: organic ligands form complexes with lanthanide ions (chelates) for
• ligand-sensitized fluorescence (=> higher absorption cross section)
• shielding lanthanide ions from contact with water (=> higher quantum yield)
Labels: fluorescent lanthanide chelates or chelate-dyed nanoparticles
Instrumentation: time-resolved fluorometer
26
Lanthanides and luminescent lanthanide chelates
C F 3
OOC F 3
O
O
C F 3
O
O
P
O
E u 3 +
Soini & Hemmilä, Clin. Chem. 1979, 25, 353
27
Lanthanides and luminescent lanthanide chelates
Lanthanide chelates:
• large Stoke's shift
• narrow emission peaks
• long fluorescence lifetime (~ 1 ms)
excitation emissionC F 3
OOC F 3
O
O
C F 3
O
O
P
O
E u 3 +
28
Principle of pulsed time-resolved fluorometer
Europium-chelate
• emission at 615 nm
• decay time ~ 1 ms
Effect of time resolution on the fluorescence
29
Four-fold labeling option with Tb, Dy, Eu and Sm
30
=> potential for multiplexing
C F 3
OOC F 3
O
O
C F 3
O
O
P
O
E u 3 +
Dissociation enhanced
lanthanide fluoroimmunoassay
- low pH
-> ion dissociates
- excess of chelate
-> fluorescent complex
Commercially available as DELFIA system
But:
• spatial information is lost
• not suitable for direct measurement from solid surface
N N N
CC
N
H
N
H
S
C
PROT
C
O O O O O O
C
O O
Eu3 +
Non-fluorescent lanthanide chelates and enhancement
31
Triton X-100 micelle
=> hydrophobic pockets
Formation of highly fluorescent chelates
Eu3+ + ß-diketone + TOPO
Eu 3+
DELFIA
32
NN
H
N
H
S
C
P R O T
N
N
C
C
C
C
O
O
O
O
O
O
O
O
E u 3 +
=> measurement from
solid-phase requires dry surface
=> spatial information is preserved
N
N
N
N
N
C
C
C
C
O
O
O
O
O
O
O
O
O
OR
R
N
H
C
S
N
H
PROT
Eu3+
Intrinsically fluorescent lanthanide chelates
=> direct measurement from
solid-phase 33
34
Digital (single-molecule) assays
=> Millions of molecules needed to reach detection limit
=> One molecule needed to reach detection limit
Serial dilution
Conventional immunoassay (analog readout)
Single-molecule immunoassay (digital readout)
35
Surpassing the traditional detection limit
CF3
OOCF3
O
O
CF3
O
O
P
O
N
H
O
N
H
O
Eu3+
PROT
PROT
30'000
107 nm
30000 europium chelates / nanoparticle
=> relatively large particles
=> very bright (easily detectable) luminescence
=> no concentration quenching
Time-resolved luminescence for digital assays
36
Nanoparticle
5A10
H117
107 nm
12 nm
Solid-phase
PSA-ACT
PSA
Eu(lll)-chelate
5A10
- the specific activity of the label can be excluded from the performance limiting factors
- multiple binding sites compensate for large diameter
Time-resolved luminescence for digital assays
37
Flash lamp
Cooled CCD
camera
Chopper
epi-fluorescentmicroscope
ex 340 nm / em 615 nm
excitation pulse 10 µsec
delay time 100 µsec
window time 600 µsec
measurement cycle 50 Hz,
30-45 sec
10x objective, NA 0.3
60x objective, NA 0.85
2 x 2 binning
Time-resolved fluorescence microscope
38
5 ng/mL
diameter~6mm
Top view on microtiter plate well
39
5 µm5 µm5 µm
SEM image
Härmä, H. et al. (2001) Clin Chem 47: 561-568
=> using time-resolved fluorescence
Observation of individual labels
40
Counting individual complexes
1 ng/mL 0.3 ng/mL
0.1 ng/mL 0.01 ng/mL
41
42
Digital immunoassay allow for the detection of single analyte molecules,
but this should not be confused with the highest analytical sensitivity
=> non-specific binding of labeled component (and
its variation) defines the actual limit of detection
non-
specific
specific
Digital (single molecule) assays
0.003 ng/mL 0 ng/mL - non-specific binding only
specific or non-specific binding? non-specifically bound label
Counting individual complexes
43
Photon-upconversion nanoparticles
(UCNPs)
44
wavelength (nm)
45
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 46
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
47
48
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
49
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
50
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
51
Microsocope Microtiter plate reader
Instruments for the detection of UCNPs
52
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
53
Microtiter plate
Mickert MJ (2019) Anal. Chem. 91, 9435
Upconversion-linked immunosorbent assay (ULISA)
54
no PSA 10 fg/mL 100 fg/mL
1 pg/mL 10 pg/mL 100 pg/mL
1 ng/mL 10 ng/mL 100 ng/mL
Anal. Chem. (2017) 89, 11825
Single UCNPs are detectable as diffraction-limited spots
Excitation power: ~640 W/cm2 55
UCNPs for digital assays
56
Detection limits of various immunoassays