C8116 Immunochemical techniques
Immunoassays II
Spring term 2024
Hans Gorris
Department of Biochemistry
April 16th, 2024
1
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
2
(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
3
4
Digital (single-molecule) assays
based on:
• life-time luminescence
• upconversion nanoparticles
• enzyme labels
• fluorescent labels
5
Microtiter plate well
11mm
7 mm
360 µl
Femtoliter well
3µm
4.5 µm
50 fl
Reduction of the volume
by a factor of 1010
Microtiter plate (96 wells) Femtoliter array (62.500 wells)
12 wells
8wells
250 wells
250wells
SEM: scale bar (10 µm)
Increasing the
number of wells
by a factor of 103
Minimizing the size of wells
6
Femtoliter arrays generated by photolithography
7
SEM images of femtoliter arrays
8
Volume 1 µM 1 nM 1 pM
(1 mm)3 1 µL 10-6 L 6×1011 6×108 6×105
(100 µm)3 1 nL 10-9 L 6×108 6×105 6×102
(10 µm)3 1 pL 10-12 L 6×105 6×102 < 1
(1 µm)3 1 fL 10-15 L 6×102 < 1
(100 nm)3 1 aL 10-18 L < 1
Poisson distribution:
with:
µ = average occupancy (0.05)
Pµ(ν) = probability of finding
exactly ν (i.e. 0,1,2,3 …)
molecules in any given well
Here:
Volume of well: 40 fL
Enzyme conc.: 1.8 pM
~5 % of the wells contain
a single enzyme molecule
( )
!n
µ
n
n
µ
µ
=
eP
Expected number of molecules
in a given volume:
On average
1 molecule per well
On average 1 molecule
in every 20th well
Isolation of single molecules in femtoliter arrays
9
Single enzyme molecule reaction
10
Observing single enzyme molecules
11
Anal. Bioanal. Chem. (2015) 407, 7443
Counting individual enzyme molecules
Linear relationship between
number of fluorescent chambers
and enzyme concentration
0.9 pM 1.8 pM
2.7 pM 3.6 pM
=> 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)
12
Surpassing the traditional detection limit
13
Nature Biotechnology (2010) 28, 595
Single-molecule ELISA on beads (Quanterix)
14
Single-molecule ELISA on beads (Quanterix)
15
=> Digitization of enzyme-linked complexes greatly increases sensitivity compared with
bulk, ensemble measurements.
Single-molecule ELISA on beads (Quanterix)
16
Digital assays: Single fluorophore counting (Singulex)
=> A separtion between antigen capture and detection
is required to avoid optical background interference.
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
17
UCNPs as background-free optical labels
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 18
UCNPs for digital assays
19
Analog vs. digital readout
specific
binding
non-specific
binding
20
Detection limits of various immunoassays
250
Anderson & Anderson (2002) Molecular & Cellular Proteomics 1, 845
Current protein blood tests (ELISA)
> 2500
10-3 M
10-12 M
Large unused potential of
diagnostic biomarkers
(human genome: 25000 genes)
Challenges:
- Limited sensitivity
- Limited dynamic range
- Imprecision of results
- Large sample size needed
We only see the tip of the iceberg
21
22
From microarrays to bead assays
23
A) Protein-protein interactions
B) Antibody array
C) Sandwich immunoassay
D) Peptide array
E) Enzyme-substrate interactions
F) Ligand-receptor interactions
G) - I) Reverse microarrays
Various applications of microarrays
24
Protocol:
(1) Add the biotinylated protein MOG1
(involved in nuclear import)
(2) Add fluorescence-labeled streptavidin
=> Binding to interaction partner GSP1
48 subarrays with 4000 different yeast proteins
Protein micorarrays (Invitrogen)
4 rows
12lines
25
PROPERTIES BLOT-LINE MICROBLOT-ARRAY
Maximum antigens per strip/well 19 44
Tests per kit 20 up to 96
Maximum capacity per strip/well 21 bands 200 spots
Sample consumption per test 30 µl 10 µl
TestLine
Detection with conjugate:
alkaline phosphatase antibodyAntigens immobilized in
a well with control spots to
1) check the presence of conjugate
2) check functionality and sensitivity
26
Microarrays: Ambient analyte assays
• Very small amounts of capture antibody do not reduce the analyte
concentration of the sample: „Ambient analyte assay“
• On a small detetction area, there is less space for non-specific binding
=> High signal density correlates with high sensitivity
27
Planar array ó Bead array
Microarray
of capture
elements
Spatially
addressable
A B C
D E F
G H I
X
Y
Measurement
X
YZ
C
A B C D E F
Z
A B C
D E F
G H I
C
A
B
D
E
F
A B C D E F
Detection Many possible
detection elements
Fluorescent labels
Fluorescence-encoded beads
In sus-
pension
28
Beads: magnetic separation
Beads can be separated by applying
an external magnetic field
=> Allows for washing steps to
remove excess reagents
29
Planar array
• Antibodies are immobilized on fixed positions on a
solid support
• Each type of analyte can be directly addressed by
its spatial location
Bead array
• Antibodies are immobilized on the surface of
beads
• An encoding strategy is required (e.g. code of
different fluorophore combinations)
Each encoded bead carries
a different type of antibody
Microarrays: Multiplexing
30
Beads: Fluorescent codes
Bead encoding by using
fluorophore combinations:
2 colors
4 intensities
Number of Codes = if - 1
42 - 1 = 15 Codes
A
Another fluorophore (color) is required
for the detection of analyte binding.
B
C
A
B
D
E
F Each encoded bead
carries a distinct
capture element
31
Readout of fluorescent codes
Flow cytometry (Luminex)
Deposition on femtoliter arrays +
fluorescence microscopy (Illumina)
C
A
B
D
E
F
Beads in suspension
=> randomly ordered=> “on the fly”
F CA
32
Planar (directed) arrays
Positional encoding of probe
elements on array
Advantages:
Simple readout
Very common
Disadvantages:
Probe molecules must be
attached to each spot
individually
Þ Batch-to-batch variation
Þ limited throughput
Suspension arrays
Sorting by flow cytometry
Randomly ordered arrays
Loading into femtoliter arrays
Advantages:
Homogeneous beads generated in a single batch
inexpensive
Only small sample volumes required
Disadvantages:
Each probe requires a specific code
=> Enables thousands of measurements in a small volume
Fluorescence-encoded beads
Three types of array formats
33
Homogeneous immunoassays
34
Heterogeneous vs. homogeneous immunoassay
Heterogeneous assay
35solid-phase
non-bound labeled antibodies are
physically separated before detection
(typically using a solid-phase)
non-competitive "sandwich" immunoassay
Homogeneous assay
36no solid-phase required!
=> mix-and-measure assay
non-competitive "sandwich" immunoassay
A
A
A
A
D
D
D
D
signal of the labeled antibody is
modulated upon binding
Examples how to modulate the detection signal
37
=> “smart” reporters for homogeneous assays
• Fluorescence resonance energy transfer (FRET)
• Luminescent oxygen channeling
• Fluorescence polarization
• Lanthanide complementation
O OHO
COOH
R
O N+
(CH3)2(H3C)2N
COOH
R
fluorescein tetramethylrhodamine
λex = 488 nm
λem = 517 nm
λex = 533 nm
λem = 565 nm
FRET
Fluorescence Resonance Energy Transfer (FRET)
Donor Acceptor
38
÷÷
ø
ö
çç
è
æ
+
=
0
1
1
R
R
E
DA
T 6
FRET: A nanoscale ruler
39
FRET: A nanoscale ruler
40
41
Detection of FRET
Excitation wavelength:
Wavelength (nm)
In solution (cuvette, microtiterplate) => Immobilization not required
Example: Assay for IgG
2 reagents:
- IgG labeled with FRET acceptor
- anti-IgG labeled with FRET donor
Principle:
IgG (analyte) and labeled IgG
compete for binding to anti-IgG;
if labeled IgG is present in large
excess (= little analyte), FRET is
efficient. Otherwise, there is little
FRET.
Steps:
Add labeled IgG and labeled anti-IgG
to the sample. That's it!
Competitive immunoassay based on FRET
42
Luminescence oxygen channeling
43
E. Ullmann et al. 1994, PNAS 91: 5427
much longer than
in FRET (<10 nm)
Luminescence oxygen channeling immunoassay
44
Fluorescence Polarization Immunoassay (FPIA)
45
Fluorescence Polarization Immunoassay (FPIA)
46
In solution (e.g in microtiter plate)
Reagents needed:
(a) labeled antigen; (b) antibody (a secondary antibody is not needed)
Reaction: competitive binding of free antigen and labeled antigen to labeled antibody.
Labeled antigen ("FP conjugate") in solution tumbles and depolarizes light.
Labeled antigen bound to antibody tumbles more slowly => less depolarized light.
Fluorescence Polarization Immunoassay (FPIA)
47
48
Immune agglutionation / precipitation
Blood type: different antigens on red blood cells
blood group A blood group B blood group AB blood group 0
blood contains antibodies
against antigen B
blood contains antibodies
against antigen A
blood contains no
antibodies against
antigen A or B
blood contains antibodies
against antigens A and B
49
50
Immune agglutination
antigen-covered microscopic particles in suspension
(e.g. bacteria, blood cells, or latex particles)
+
Specific immune serum / antibodies
II
V
cross-linking forms large aggregates
that are not stable in suspension
(agglutination)
II
V
visible sedimentation
Advantages:
Cheap, easy, very sensitive,
but semi-quantitative
51
Immune agglutination: blood type
Determination of blood type in microtiter plate
Evaluation:
positive: agglutination, bead formation
negative: erythrocytes remain in suspension
(homogeneous red fluid)
Test series blood
group
no agglutinationagglutination
52
Immunoprecipitation
soluble antigen
+
specific antibodies
II
V
cross-linking forms insoluble antibodyantigen
complexes
(=> remember: at least 2 or
more epitopes required)
II
V
visible sedimentation
antigen concentration
Amountofprecipiatedantibody
zone of
antibody
excess
zone of antigen
excess
equivalencezone
free antibody
complex-bound ab
free antigen
complex-bound ag
53
Nephelometry
Time (min)
Degreeofturbidity(%)
high antigen
concentration
low antigen
concentration
54
Classification of immunoprecipitation systems
antigen
antibody
1% agarose
1-dimensional diffusion
2-dimensional diffusion
A) Oudin
B) Oakley / Fulthorpe
C) Mancini
D) Ouchterlony
simple diffusion
double diffusion
1-dimensional immune diffusion (Oudin)
antigen (A or A,B)
antibody
agarose
soluble immune complexes
soluble immune complexes + antibody A
Immune precipitation disc
Contact area between ag and ab
55
56
2-dimensional immune diffusion (Mancini)
Area
Antigen concentration
1-4: increasing
antigen concentration
gel contains antiserum
57
2-dimensional immune diffusion (Ouchterlony)
Non-
identical
Identical
Schemes of immune precipitation patterns
Photograph:
4 holes punched out
in agarose gel
immune
precipitates
anti-
gens
anti-
bodies
58
Immune diffusion
complex
antigen sample
polyclonal
antiserum
Problem:
patterns cannot be resolved
vertical
gel
Protein electrophoresis
Migration of proteins/antigens:
v = q * E / fc
59
slab gel
(prevents convective mixing and serves as molecular sieve)
agarose
(more common for DNA)
polyacrylamide (PAGE)
larger pore size smaller
(1%: 150 nm) (5 nm)
=> both types of matrices are electrically non-conductive
Matrices for protein electrophoresis
60
61
Immune electrophoresis
Separation of proteins in electric field
groove for
antibody sample
62
Cross electrophoresis
Separation of proteins in electric field
groove for
antibody sample
Cross electrophoresis
=> 2nd electrophoresis into
agarose gel containg antibody sample
Immunofixation
-
+
Electricfield
Different proteins
1) electrophoresis in e.g. agarose gel
2) diffusion from the agarose gel onto
a cellulose acetate membrane (does not bind proteins)
3) immune complexes precipitate on membrane and are not washed out
63
64
Guest lecture: next week
Prof. Tero Soukka
University of Turku, Finland
Department of Life Technologies/Biotechnology
1 pm: Evolution of lanthanide-based
labels for immunoassays
2 pm: Research talk open for all
65
Thank you for your attention