Surface plasmon resonance (SPR) applications in immunology
Biosensors and bioassays
Data generation and analysis
•
Experimental design
•
Information extracted from data
Antibodies for therapeutic and biotechnological applications
•
Concentration determination
•
Class determination
•
Binding kinetics
•
Binding stoichiometry
•
Epitope determination
•
Screening / ranking
Danièle Altschuh
[email protected]
http://biocapteurs.u-strasbg.fr/
ESBS, Parc d'Innovation, Boulevard S. Brant
BP 10413, 67412 ILLKIRCH Cedex, France
Definition of a biosensor
A biosensor is an analytical device comprising two elements
in spatial proximity:
A biological recognition element able to interact
specifically with a target
A transducer able to convert the recognition event into a
measurable signal
Biosensors and bioassays
A) Bioassay
Interaction
Separation
Detection
B) Biosensor
Interaction = signal (detection)
Recognition unit
Target
Recognition elements
• Proteins (Enzymes, antibodies, receptors, antigens)
• Nucleic acids (Ribozymes, Aptamers, Hybridization sequences)
• Cells
• ……….
Transducers
• Electrochemical
• Thermometric
• Surface Plasmon Resonance
• Interferometry
• …….
Hoa XD, Kirk AG, Tabrizian M. (2007)
Towards integrated and sensitive surface plasmon resonance biosensors: a review of recent progress.
Biosens Bioelectron. 2007 Sep 30;23(2):151-60. Epub 2007 Jul 20. Review.
Instrumentation (non-exhaustive)
SPR
Rich RL, Myszka DG.
Survey of the year 2006 commercial optical biosensor literature.
J Mol Recognit. 2007 Sep-Oct;20(5):300-66.
(Horiba)
SensíQ® (Nomadics)
……
www.discoversensiq.com/products/sensiq/
QCM
Cooper MA, Singleton VT.
A survey of the 2001 to 2005 quartz crystal microbalance biosensor literature: applications of
acoustic physics to the analysis of biomolecular interactions.
J Mol Recognit. 2007 May-Jun;20(3):154-84. Review.
Biacore 3000
Biacore T100
GE Healthcare Biacore
http://www.biacore.com
Principle of the BIACORE technology (GE Healthcare - Biacore, Uppsala, Sweden)
BIACORE instruments are optical biosensors based on the phenomenon of surface plasmon resonance (SPR).
1
Intensity
prism
glass
gold
Sensor Surface
1
Angle
Flow
2
2
RU
Sensorgram
2
prism
Sensor Surface
glass
gold
1
Flow
Time
The variation in SPR signal (position of the resonance angle) is expressed in arbitrary units called resonance unit : RU
It is expressed as a function of time. The resulting graph is called a sensorgram.
1000 RU = 1ng protein/mm2
Biacore® SPR technology: http://www.biacore.com/technology/core.lasso
The sensor surface
Ligand
Carboxylated dextran
(non cross-linked)
Linker
Gold
Glass
Properties:
* Aqueous environnement (hydrogel containing 97-98% water)
* Mobility (chains are not cross-linked)
* Efficient use of the evanescent field (thickness about 100 nm)
* Increased sensitivity (more coupling sites than on a flat surface)
* Allows covalent coupling (through carboxyl groups)
The phases of a sensorgram
RU
10
Time (s)
Response (RU)
Analyte injection
phase
Post-injection
phase
Regeneration
17000
16000
15000
14000
0
120
Time (s)
240
360
Simulated kinetics for Ka = ka/kd = 109 M-1
ka (M-1.sec-1)
kd (sec-1)
Ka (M-1)
104
105
106
107
10-5
10-4
10-3
10-2
109
109
109
109
Response(RU)
250
200
150
100
50
0
0
120
240
360
480
600
Time (seconds)
(PM 50000, Rmax 200 Rus, Flow 10 µl/mn, A= 50 nM)
Surface plasmon resonance (SPR) applications in immunology
Biosensors and bioassays
Data generation and analysis
•
Experimental design
•
Information extracted from data
Antibodies for therapeutic and biotechnological applications
•
Concentration determination
•
Class determination
•
Binding kinetics
•
Binding stoichiometry
•
Epitope determination
•
Screening / ranking
Experimental design
- Immobilization of the ligand (recognition element, receptor)
- Reference surfaces
- Regeneration
Active (ligand) surface
* Covalent
- amine coupling
- thiol coupling
Analyte
Ligand
* Captured ligand
- streptavidin/biotin
- NTA/His-tag
- hydrophobic surfaces/lipids
- anti-antibody/antibody
- anti-GST/GST(fusion proteins)
- anti-peptide/peptide (fusion proteins)
Analyte
Ligand
Capture
Reference surface
Injection
RU
17500
Post-injection
Régénération
17000
16500
16000
15500
Response
15000
14500
14000
13500
0
40
80
120
160
200
240
280
320
Time
RU
1600
Injection
Post-injection
1400
1200
1000
800
Response
600
400
200
0
-200
0
50
100
Time
150
200
250
s
360
400
s
Reference substraction
Refererence surface
Active surface
Injection phase
Injection phase
Active - reference
Experimental conditions
[temperature, solvent viscosity (diffusion), flow rate, A0, Lfree]
Kinetic conditions
Mass transport conditions
A0 (C)
A0 (C)
=
=
Asurf
Asurf
Affinity parameters
A0
km
km
Asurf + L
Quantification of the analyte
ka
kd
AL
A0
km
km
Asurf + L
ka
kd
Goldstein, B., Coombs, D., He, X., Pineda, A. R., and Wofsy, C. (1999) The influence of transport on the kinetics of binding to surface
receptors: application to cells and BIAcore, J Mol Recognit 12, 293-9
Karlsson, R., Roos, H., Fägerstam, L., and Persson, B. (1994) Kinetic and concentration analysis using BIA technology, Methods : A
Companion to Methods in Enzymology 6, 99-110
AL
Kinetic conditions :
Affinity, kinetic parameters
Mass transport conditions :
Active analyte concentration
30
1000
Rmax = 50 RUs, [A0] = 1 & 10 nM
Rmax = 5000 RUs, [A0] = 1 & 10 nM
750
20
500
10
250
0
0
60
120
180
240
300
dR
= ka ∗ A0 ∗ (Rmax − R) − kd ∗ R
dt
R=
(
ka ∗ A0 ∗ Rmax ∗ 1− e
ka ∗ A0 + kd
−(ka ∗A0 + kd )t
0
0
60
120
180
240
300
dR /dt ≈ kt ∗ A0
)
ka = 10+6 M-1 s-1 kd = 10-2 s-1 Ka = 10+8 M-1
MM= 25000, Fl=30 microl/mn
Information extracted from SPR data
•
Detection of binding (low affinity), ranking of binders
•
Quantitative characterization of binding
- Affinity (Ka, Kd), kinetic (kon, koff), thermodynamic (∆H, T∆S) parameters
- Binding stoichiometry
•
Molecular properties
- Active concentration
- Multimerisation - Aggregation - Homogeneity
Détection d’une interaction
- Criblage
- classement
Anticorps
Composés chimiques
MW 400 - 600 da
MW 150.000 da
1 min injection at 20µM
(6 -40 RU)
2 min injection
Injection
Injection
Post-injection
Response (RU)
25
20
1,2
4
15
3
10
5
6
5
0
-5
0
50
100
Time (s)
150
200
Post-injection
Affinity (Ka, Kd) and kinetic (kon, koff) parameters
scFv LR28B4
RU
(31 - 500nM)
40
30
scFvs sur une surface gastrine
20
10
0
0
50
100
150
Time
200
250
s
ka (1/Ms) kd (1/s) Rmax (RU) KA (1/M) KD (M) Chi2
2.76e4
5.79e-4
54.8
4.77e7
2.1e-8 1.36
R =
ka . C . Rmax
ka . C + kd
x (1 - e - (ka . C + kd ) . t )
Measurements of active analyte concentrations in total mass transport conditions
Karlsson R, Roos H, Fägerstam L, Persson B. Kinetic and concentration analysis using BIA technology. 1994. Methods 6, 97-108.
9000
80 nM
5000
Calibration curve
40 nM
3000
20 nM
10 nM
5 nM
1000
-1000
60
210
360
510
660
Time (sec)
Binding rate (RU/sec)
Response (RU)
7000
10 20
Simulated sensorgrams (high ligand density)
ka
105 M-1.sec-1
kd
10-3 sec-1
Rmax
50.000 RU
PM
25.000 da
40
Analyte concentration (nM)
[A]
variable
80
Calibration Free Concentration Analysis (CFCA)
Biacore T100 evaluation software 2.0.1. (GE-Healthcare Biacore)
Curve
Sample
Dilution
factor
Meas.Conc
(M)
Calc. Conc
(M)
Fc=4-3
scFv1F4-M
600
1.686E-09
1.012E-06
Aggregates / multimers (Binding stoichiometry )
Rmax (analyte)
RLigand
Rmax
RLigand
Rmax
RLigand
Rmax
RLigand
Rmax
RLigand
Rmax
RLigand
=
=
=
MMAnalyte
MMLigand
2 x MMAnalyte
MMLigand
MMAnalyte
MMLigand
Bivalent analyte binding model to fit kinetic data
Langmuir and complex kinetics
Langmuir binding
Bivalent binding model
No fit
Réponse (RU)
16
koff = 0,001 s-1 Ka = 108 M-1
[A]=10 nM (30 kda)
12
Rmax = 100 RU
8
koff = 0,01 s-1 Ka = 107 M--1
4
koff = 0,1 s-1 Ka = 106 M-1
koff = 1 s-1 Ka = 105 M-1
0
0
100
200
300
400
Réponse (RU)
Temps (s)
12
[A]=10 nM (30 kda)
Rmax = 10000 RU
8
Rmax
4
Ka = 105 M-1
Rmax = 1000 RU
0
0
100
200
Temps (s)
300
400
Réponse (RU)
60
[A] = 10000 nM
[A]
40
Rmax = 100 RU
20
[A] = 1000 nM
Ka = 105 M-1
[A] = 100 nM
0
0
100
200
Temps (s)
300
400
Data interpretation
Lower concentration, stronger binding
SPR
Response (RU)
Conventional assay
Rmax
Time (sec)
Response (RU)
Higher concentration, weaker binding
Rmax
Time (sec)
Aggregates
Response (RU)
Rmax
Time (sec)
Data generation and analysis
De Crescenzo G., Boucher C., Durocher Y. and Jolicoeur M. (2008).
Kinetic Characterization by Surface Plasmon Resonance-Based Biosensors: Principle and
Emerging Trends
Cellular and Molecular Bioengineering Volume 1, Number 4 / 204-215.
Karlsson, R., and Fält, A. (1997)
Experimental design for kinetic analysis of protein-protein interactions with surface plasmon
resonance biosensors
J Immunol Methods 200, 121-33.
Myszka, D. G. (1999)
Improving biosensor analysis
J Mol Recognit 12, 279-84
Rich RL, Myszka DG.
Survey of the year 2007 commercial optical biosensor literature
J Mol Recognit. 2008 Nov-Dec;21(6):355-400. Review.
Surface plasmon resonance (SPR) applications in immunology
Biosensors and bioassays
Data generation and analysis
•
Experimental design
•
Information extracted from data
Antibodies for therapeutic and biotechnological applications
•
Concentration determination
•
Class determination
•
Binding kinetics
•
Binding stoichiometry
•
Epitope determination
•
Screening / ranking
Antibodies for therapeutic and biotechnological applications
Antigen
Ab repertoire
(in vivo)
Ab library
(in vitro)
Immunization
Serum
Polyclonal antibodies
- Detection
Hybridoma cell
Monoclonal antibodies
Recombinant antibodies
- Selection
- Characterization of properties
- Characterization / optimization of properties
Antibody design criteria: affinity, selectivity, class, expression level, stability….
Assay objectives
• Screening/ranking
• Concentration
• Binding parameters (kon, koff, KD)
• Selectivity
• Subclass determination
• Epitope identification
Samples to characterize
• Therapeutic antibodies
- monoclonal for passive immunization
- polyclonal (serum) for passive immunization
- polyclonal (serum) in active immunization
• Unwanted serum antibodies (drug immunogenicity)
• Antibodies for biotechnological applications
- monoclonal
- polyclonal (serum)
Concentration determination
Chavane N, Jacquemart R, Hoemann CD, Jolicoeur M, De Crescenzo G. (2008 )
At-line quantification of bioactive antibody in bioreactor by surface plasmon
resonance using epitope detection.
Anal Biochem. 378:158-65.
Aim
to monitor the concentration of bio-active antibody (unpurified) secreted during bioreactor culture
mAb (unpurified sample)
Antigen (peptide)
Mass transport conditions (Rmax 12.000 - 40.000 RU)
- High antigen immobilization level (400-3000 RU)
- (Low) flow rate (20 µl/mn)
- Sample injection (1/20 dilution in running buffer)
- Short injection and post-injection phases
- Regeneration (HCl-glycine pH 2.0)
dR /dt ≈ kt ∗ A0
Chavane et al. (2008 ) Anal Biochem. 378:158-65.
mAb poduced by
hybridoma cell line
Subclass determination
van Remoortere A, van Dam GJ, Hokke CH, van den Eijnden DH, van Die I, Deelder AM. 2001
Profiles of immunoglobulin M (IgM) and IgG antibodies against defined carbohydrate epitopes in
sera of Schistosoma-infected individuals determined by surface plasmon resonance.
Infect Immun. 69:2396-401.
Aims
- to monitor the presence of serum antibodies to carbohydrate epitopes in
the sera of individuals infected with parasites (Schistosama)
- to define the IgG and IgM subclass distribution of the antibodies
Class specific Ab
Conditions
« Loose »
- antigen (glycoprotein ~4000 RU)
- flow rate 5 µl/mn
- sera diluted 1/40 in running buffer
- class specific ab diluted 1/100 in running buffer
- 2 min injection and post-injection phases
- regeneration 100 mM HCl
Serum Ab
Antigen
van Remoortere A et al. (2001) Infect Immun. 69:2396-401.
Serum ab
Anti IgM
Anti IgG
Binding kinetics
Barderas R, Shochat S, Timmerman P, Hollestelle MJ, Martínez-Torrecuadrada1 JL, Höppener JWM,
Altschuh D, Meloen R, Casal JI (2007). Designing antibodies for the inhibition of gastrin activity in
tumoral cell lines. Int. J. Cancer 122: 2351-9
V
V
Aims
- to characterize the gastrin binding properties of antibody fragments
- to select candidates for cellular assay
H
1
CH
L
CL
CH2
CH3
Kinetic conditions (Rmax 100 - 1000 RU)
- Low antigen immobilisation level (10 - 130 RU))
- (High) flow rate (30 µl/mn)
- Injection of a range of Ab concentrations
- 1mn injection and 5mn post-injection phases
Recombinant
antibody fragment
Antigen (gastrin)
R=
(
ka ∗ A0 ∗ Rmax ∗ 1− e
ka ∗ A0 + kd
−(ka ∗A0 + kd )t
)
scFv LR28B4
RU
RU (31 - 500nM)
40
400
30
300
20
200
10
100
scFv MBE7
(31- 1000 nM)
0
0
0
50
100
150
200
Time
0
250
50
100
150
200
ka (1/Ms) kd (1/s) Rmax (RU) KA (1/M) KD (M) Chi2
2.76e4
5.79e-4
54.8
4.77e7
2.1e-8 1.36
ka (1/Ms) kd (1/s) Rmax (RU)
1.29e5
0.108
575
s
KA (1/M) KD (M) Chi2
1.19e6 8.38e-7 14.3
?
RU
scFv 119EB1
20
(69 -1100 nM)
40
Response
RU
25
15
10
250
Time
s
Rmax calc =94
RU
20
0
5
-20
0
0
50
100
150
200
Time
ka1 (1/Ms) kd1 (1/s) ka2 (1/RUs) kd2 (1/s) Rmax (RU)
1.11e5
0.0526
7.4e-4
2.46e-3
44.3
250
0
s
Chi2
0.5
ka1 (1/Ms) kd1 (1/s)
518
0.322
100
Time
ka2 (1/RUs) kd2 (1/s)
2.69e-5
2.95e-3
200
300
Rmax (RU)
338
s
Chi2
0.613
Identification d’un épitope
- Cartographie d’épitope (Epitope mapping)
- Analyse mutationelle
- Inhibition ou compétition
Epitope identification
Rauffer N, Zeder-Lutz G, Wenger R, Van Regenmortel MH, Altschuh D
Structure-activity relationships for the interaction between cyclosporin A derivatives and
the Fab fragment of a monoclonal antibody.
Mol Immunol. 1994 Aug;31(12):913-22.
Aim
to identify the functional (versus structural) epitope
Affinity in solution (mass transport conditions)
Use a ligand surface to calibrate the free antibody in antibody-antigen mixtures
Recombinant
antibody fragment
+
Cyclosporin analog
Antigen (cyclosporin)
f: Ratio bound/total Fab
d: free Fab concentration
Crystallographic structure of
the complex between Fab R45-45-11 and cyclosporin
H3: Thr H95, Leu H97,
Gly H100C, Asn H100D,
Tyr H100E, Pro H100F,
Trp H100J
L1: Tyr L32
L2: Tyr L50
3
L2: Gly L91
Ser L92
5
11
H1: Tyr H33
Tyr H35
H2: Phe H50
Asn H52A
Altschuh D, Vix O, Rees B, Thierry JC. 1992. Science 256 : 92-4
Vix O, Rees B, Thierry JC, Altschuh D. 1993. Proteins 15: 339-48.
Cyclosporin
ELISA versus SPR
Effect on binding of
cyclosporin modifications
Cyclosporin
2
Cs residue
3
ELISA data
(Quesniaux et al., 1987)
Biacore data
(Rauffer et al., 1994)
DDG values
1
4
11
5
6
10
7
9
8
MeBmt 1
Abu 2
Sar 3
MeLeu 4
Val 5
MeLeu 6
Ala 7
+
+++
+++
+++
+++
-
not modifi ed
- 0.2 to + 1.2
≥ + 3.5
+ 1.3 to + 2.2
≥ + 4.8
+ 0.9
+ 0.9
D-Ala-8
MeLeu 9
MeLeu 10
MeVal 11
+
+++
+ 0.6
+ 0.04 to + 2.1
+ 1.0 to + 3.0
≥ + 3.9
Quesniaux VF, Tees R, Schreier MH, Wenger RM, Van Regenmortel MH. 1987b. Fine specificity and cross-reactivity of
monoclonal antibodies to cyclosporine. Mol. Immunol. 24: 1159-68.
Rauffer N, Zeder-Lutz G, Wenger R, Van Regenmortel MH, Altschuh D. 1994. Structure-activity relationships for the interaction
between cyclosporin A derivatives and the Fab fragment of a monoclonal antibody. Mol. Immunol. 31: 913-22
Structure - binding activity relationship
Antigen
X
Structural data
Y
CsA residue
Antigen
X
Antibody
Y
MeBmt 1
Abu2
Sar 3
MeLeu 4
Val 5
MeLeu 6
Ala 7
D-Ala-8
MeLeu 9
MeLeu 10
MeVal 11
Area
Fraction
buried (%) buried (%)
10.5
10.2
11.9
19.8
5.4
8.1
0
0.9
14.8
2.1
16.3
32.1
64.1
98.1
67.6
81.8
30.4
0
7.3
59.1
18.1
93.8
Biacore data
∆∆G values
not modified
- 0.2 to + 1.2
≥ + 3.5
+ 1.3to +2.2
≥ + 4.8
+ 0.9
+ 0.9
+ 0.6
+ 0.04to +2.1
+ 1.0to +3.0
≥ + 3.9
The importance of a residue for binding activity correlates well with the fraction of its
surface that becomes burried upon complex formation (not with the area buried)
Structure - Binding activity Relationship
Is it possible to associate energy values with the contacts observed in the crystal structure ?
Experimental
Theoretical
Atoms lost
∆∆G (kcal/mol)
∆∆G (kcal/mol)
Ratio
Ala-2-Cs
Cγ
1.2
1.3
0.9
MeVal-4-Cs
Cβ
2.2
2.4
0.9
Leu-4-Cs
CN
1.5
0.3
5.0
MeVal-6-Cs
Cβ
0.9
0.8
1.1
Leu-10-Cs
CN
3.0
0.1
30.0
Cγ1,Cγ2
≥3.9
2.2
≥1.8
Analog
MeAla-11-Cs
Experimental : ∆∆G = -RT ln [Ka(analog)/(Ka (CsA)
Theoretical : ∆∆G = area buried x 24 cal/A2
(Chothia, 1974, Nature 248:338-339)
Binding stoichiometry
Dennehy KM, Elias F, Zeder-Lutz G, Ding X, Zigan P, Altschuh D, Fred Lühder F & Hünig T (2006)
Monovalency of CD28 maintains the antigen-dependence of T cell co-stimulatory responses.
J. Immunol. 259: 77-86
Aim
To investigate if the bivalent analyte binds monovalently or bivalently to the
bivalent ligand
Conditions that allow surface saturation
- Low ligand immobilisation level (~50-60 RU)
- Injection of a saturating analyte concentration (20 or -1000 nM))
- Repeated analyte injections
CD28Ig
(Co-stimulatory TCR)
Ab
Dennehy KM et al. (2006) J. Immunol. 259: 77-86
Screening/ranking
Leonard P, Säfsten P, Hearty S, McDonnell B, Finlay W, O'Kennedy R.
High throughput ranking of recombinant avian scFv antibody fragments from crude lysates using the Biacore A100.
J Immunol Methods. 2007 Jun 30;323(2):172-9
Wassaf D, Kuang G, Kopacz K, Wu QL, Nguyen Q, Toews M, Cosic J, Jacques J, Wiltshire S, Lambert J,
Pazmany CC, Hogan S, Ladner RC, Nixon AE, Sexton DJ. (2006).
High-throughput affinity ranking of antibodies using surface plasmon resonance microarrays.
Anal Biochem. Apr 15;351(2):241-53
Aim
- Identify highest affinity binders from antibody libraries
- Identify active site binders (inhibition)
Kinetic conditions
- Low ligand immobilisation level
- 5-7 mn post-injection phase
- Regeneration (10 mM NaOH / no))
Leonard P, Säfsten P, Hearty S, McDonnell B, Finlay W, O'Kennedy R.
High throughput ranking of recombinant avian scFv antibody fragments from crude lysates using the Biacore A100.
J Immunol Methods. 2007 Jun 30;323(2):172-9
0 - 217 RU
(30 nM)
C reactive protein
0 - 200 RU
scFv to screen
(expression levels)
2000 RU
(50 µg/ml)
Biotinylated
anti HA-tag antibody
Streptavidine
Control surfaces
96 scFvs / 6 hours
8/cycle
Leonard P et al. (2007). J Immunol Methods. 323(2):172-9
Wassaf D, Kuang G, Kopacz K, Wu QL, Nguyen Q, Toews M, Cosic J, Jacques J, Wiltshire S, Lambert J,
Pazmany CC, Hogan S, Ladner RC, Nixon AE, Sexton DJ. (2006).
High-throughput affinity ranking of antibodies using surface plasmon resonance microarrays.
Anal Biochem. Apr 15;351(2):241-53
Cell-based SPR biosensor (Biacore 3000)
30-113 RU
(4-250 nM)
Fab
30-113 RU
Biotinylated ag
(human tissue kallikrein)
Array-based SPR biosensor (Flexchip)
(100 nM)
Wassaf D et al. (2006) Anal Biochem. Apr 15;351(2):241-53
Affinity ranking
hK1 100 nM
Inhibition
- hK1
- hK1 + aprotinin
Wassaf D et al. (2006) Anal Biochem. Apr 15;351(2):241-53
KD = koff / kon
Antibodies as biosensor recognition elements
Chambers JP, Arulanandam BP, Matta LL, Weis A, Valdes JJ.
Biosensor recognition elements
Curr Issues Mol Biol. 2008;10(1-2):1-12. Review.
Conroy PJ, Hearty S, Leonard P, O'Kennedy RJ.
Antibody production, design and use for biosensor-based applications.
Semin Cell Dev Biol. 2009 Feb;20(1):10-26
Antibodies as therapeutic molecules
Tabrizi MA, Bornstein GG, Klakamp SL, Drake A, Knight R, Roskos L. (2009)
Translational strategies for development of monoclonal antibodies from discovery to the clinic.
Drug Discov Today. 14:298-305. Review.
Selection of the “best” antibody for each application requires high quality data, recorded using
high information content technologies, such as SPR
Ligler FS.
Perspective on optical biosensors and integrated sensor systems.
Anal Chem. 2009 Jan 15;81(2):519-26. Review
Luong JH, Male KB, Glennon JD. (2008).
Biosensor technology: technology push versus market pull.
Biotechnol Adv. Sep-Oct;26(5):492-500. Review
Multi-analyte surface plasmon resonance biosensing.
Homola J, Vaisocherová H, Dostálek J, Piliarik M.
Methods. 2005 Sep;37(1):26-36. Epub 2005 Sep 30. Review.
The SPR transducer incorporates a thin metal film which
supports a special mode of electromagnetic field—a surface
plasmon polariton (SPP)—sometimes referred as to a surface
plasma wave. The SPP propagates along the surface of the
metal film and the intensity of its electromagnetic field
exponentially decays from the metal surface into the adjacent
medium. The most commonly used metal is gold due to its
chemical stability.
A change in the refractive index due to the binding of analyte
molecules to biomolecular recognition elements immobilized on
the metal surface results in a change in the propagation constant
of the SPP. Surface plasmon resonance biosensors take
advantage of this phenomenon and measure changes in the
propagation constant of the SPP to determine changes in the
amount of bound analyte and subsequently the concentration of
analyte in a sample.
Changes in the propagation constant of the SPP are determined
by measuring one of the characteristics of the light wave that
excites the SPP. On the basis of the characteristic of the light
wave which is measured, SPR sensors are classified as sensors
with angular, wavelength, intensity, phase, and polarization
modulations.
Surface plasmon resonance sensors for detection of chemical and biological species.
Homola J.
Chem Rev. 2008 Feb;108(2):462-93. Epub 2008 Jan 30. Review.
Towards integrated and sensitive surface plasmon resonance biosensors: a review of recent
progress.
Hoa XD, Kirk AG, Tabrizian M.
Biosens Bioelectron. 2007 Sep 30;23(2):151-60. Epub 2007 Jul 20. Review.
Multi-analyte surface plasmon resonance biosensing.
Homola J, Vaisocherová H, Dostálek J, Piliarik M.
Methods. 2005 Sep;37(1):26-36. Epub 2005 Sep 30. Review.
Looking towards label-free biomolecular interaction analysis in a high-throughput format: a review
of new surface plasmon resonance technologies.
Boozer C, Kim G, Cong S, Guan H, Londergan T.
Curr Opin Biotechnol. 2006 Aug;17(4):400-5. Epub 2006 Jul 11. Review.
The two compartment model
External
compartment
A0
Internal
compartiment
Asurf
Lfree
Surface
AL
ktransp
A0
ka
Asurf + Lfree
ktransp
AL
kd
ktransp : mass transport coefficient
In Biacore
3
D2 x fl
km = 0.98 x
H2 x W x 0.3 x L
D = analyte diffusion constant
H, W, L = flow cell height, width, length
fl = volumetric flow
Caractérisation quantitative de l’interaction
- Influence de l’environnement sur kon, koff
cofacteurs ou d’additifs…)
(pH, température, présence de
Dejaegere A, Choulier L, Lafont V, De Genst E & Altschuh D. (2005) Variations in AntigenAntibody Association Kinetics as a Function of pH and Salt Concentration: A QSAR and
Molecular Modeling Study. Biochemistry 44: 14409-14418
RU
60
RU
100
pH 6, 20°C, 50 mM NaCl
pH 6, 20°C, 500 mM NaCl
52
80
44
36
60
28
40
12
Response
Response
20
4
-4
20
0
-12
-50
2
0
50
100
Time
150
200
250
s
-50
0
50
100
Time
150
200
250
s