- Email: [email protected]
Surface plasmon resonance for detection and measurement of antibody-antigen affinity and kinetics
Surface
plasmon
measurement
resonance
for detection
of antibody-antigen
affinity
and
and kinetics
Magnus Malmqvist Pharmacia Biosensor
Kinetic and affinity information by analysis based
directly
of adsorption
analytical
AB, Uppsala, Sweden
on biospecific
of analyte
interaction
to immobilized
system. The analysis can be performed
using culture
supernatants.
Current
Opinion
valuable
1993, 5:282-286
lized ligand on the same spot and at the same time it occurs. The adsorption can be followed by SPR in realtime without labels and was first used for detection of antigen-antibody interaction by Liedberg et al. [ 11.
For the understanding of the structure-function relationship in an antibody-antigen interaction and for the technical use of antibodies, the characteristic properties of the interaction must be known. Accurate measurements of physical-chemical parameters, such as rate constants and affinity, are important as well as determination of binding epitopes. The practical use of antibodies for diagnostic purposes is well established, but new rapid techniques put higher demands on kinetic properties of the immunoreagents. Therapeutic use of antibodies also requires a more detailed understanding of the affinity and kinetics of antibodies, not least when human antibody fragments are to be used. The development of recombinant antibodies has provided tools to generate and modify properties of human antibody fragments. This development must be combined with rapid and reliable techniques to measure binding activity of generated molecules. Surface sensitive optical techniques, such as surface plasmon resonance (SPR), in combination with surface chemistry and accurate liquid handling, can be used for the rapid generation of kinetic and affinity information. Detection of the interactions without the use of label is applicable to any ligand-analyte pair and kinetic information of native molecules can be generated without purification of the components. This new technology has been particularly useful for detection and quantification of antigen-antibody interactions and is now also used for analysis of DNA protein and receptor-ligand pairs.
detection
a sensor
interactions.
in Immunology
Introduction
antibody-antigen
on
free of label and
It was found to be particularly
for analysis of antibody-antigen
SPR for the label-free
can be generated ligand
A system for flow of analyte samples over a surface with immobilized ligand and detection of the adsorbed analyte by SPR has been described by Jonsson et al. [2**,3*] The practical use of SPR was not possible until a system was commercially available under the name of BIAcoreR (Pharmacia Biosensor, Uppsala, Sweden) (Fig. 1). This system combines a detector, a sensor chip for immobilization of ligands and an integrated liquid handling fluidics for transport of samples and reagents to the place of interaction. The adsorption of anylate from the continuous flow of sample to the covalently immobilized ligand is measured as a function of time. After the sample pulse has passed the probed surface, the biospecific complex dissociates, measured as a decrease in response signal. The response [resonance signal (RU)] from the instru ment as a function of time is called a sensogram (Fig. 2). Most published work on kinetic and afhnity measurements based on surface sensitive techniques has been done using this instrument system. The interface between the liquid phase and the sensor chip has to fulfil several criteria regarding non-specific adsorption, immobilization procedures and stability. Sofas and Johnsson [4] have developed a surface consisting of a gold film covered by a layer of carboxylated dextran. Both large and small molecules can be covalently coupled to carboxyl groups on the sensor chip through amine, thiol and aldehyde functional groups [ 5**,6*I.
of
The matrix, which contains 2-3 per cent dextran is hydrophilic and coupled to the thin gold film through a linker layer without any crosslinking of the polymer [4]. This gives the matrix a flexible, hydrophilic, open structure with a high capacity for protein adsorption
interaction
The basic idea for any surface sensitive detector is to measure the adsorption of the analyte to an immobi-
Abbreviations ELISA-enzyme-linked K,association
equilibrium
immunosorbent constant;
w-single
282
assay; HIV-l-human
k,-association
chain; WI-surface
@
Current
rate constant;
immunodeficiency
kc
dissociation
plasmon
resonance;
TM-tobacco
Biology
Ltd ISSN 0952-7915
virus type 1; IL-interleukin;
rate constant; mosaic
mA&monoclonal virus.
antibody;
Surface
Light source
Photo detector array
dasmon
resonance
Malmavist
and affinity constants [ 11**,12*,13=]. The association rate constant (kJ can readily be obtained from a series of analyte concentrations. However, it is important to have an accurately determined concentration of active analyte for k, determinations. This can be done with BIAcore (R Karlsson, unpublished data) working under diflusion limited conditions and using a high surface concentration of ligand. The c&fusion rate to the surface is directly proportional to concentration so both diffusion and activity of the analyte are taken into consideration. For a k, of 106 M-1 s-1 the resolution of the instrument starts to be limiting as a very low concentration of analyte must be used.
Resonance signal [RUI
Fig 1. Real time SPR monitor. The figure shows the optical configuration together with the sensor chip and flow channels. The polarized light illuminates the sensor chip at the point where the biospecific interaction takes place and is reflected to the detector array. Through surface plasmon resonance, a dark spot can be seen on the detector for one angle of incidence. This angle for extinction of light is changed when analyte is adsorbed on the immobilized ligand on the sensor chip and is indicated on the figure as another dark line to the detector. [7*,8]. Both tissue culture media and bacterial broth give extremely low non-specific background on immobilized ligands [PI.
In order to define the liquid flow characteristics in the thin-layer channels and direct the liquid to the right place, a new type of miniaturized fluidic cartridge was created [lo-]. Regeneration conditions for the elution of all bound material have to be established and the activity of the immobilized ligand maintained. Most antibody systems need regeneration but there are exceptions. It was found that useful reagents for regeneration are strong acids, such as hydrochloric and phosphoric acid [p] The conditions have to be checked for every new immobilized l&and, analyte and regeneration agent. Kinetic and affinity
measurements
New techniques and methods for BIAcore as well as the interpretation of data are developing rapidly. More correlation data between other methods and techniques are needed as few results have been published. Results obtained from BIAcore should thus at present be regarded as estimates of alhnity and kinetic constants. The association phase of the so called sensorgram (Fig. 2) is a function of the kinetic properties of the reaction, as long as the mass transport of analyte to the immobilized ligand is not a limiting factor in the reaction. Flow rate, flow cell properties and diEusion coefficient determine the mass transport of analyte [lo*]. The law of mass action describing the reaction between analyte and l&and can be rewritten in terms of response signals derived from a flow system for calculation of kinetic
Time(s)
Fig 2. A sensorgram showing the response from the SPR detector [Resonance signal (RU)] when adsorption of an analyte to the immobilized ligand takes place. From the adsorption part of the sensorgram association rate constant can be determined. After the sample pulse has passed, dissociation of the complex on the surface begins, which can be used for calculation of dissociation rate constants.
The dissociation rate constant (b> can be determined from the association part of the sensorgram. However, in practice it is only valid for extremely high values. Sta tistical analysis based on non-linear regression analysis of the association part of a sensogram has the potential to give better resolution of kinetic properties. The b can be ascertained by measuring the dissociation of bound analyte in buffer flow after the sample pulse has passed the probed surface. However, it is important to ensure that analyte is free of aggregates to avoid mixed interactions [ 14**,15**], which can cause disturbances in the evaluation. Rebinding of analyte to unoccupied lig ands should also be avoided during the dissociation from immobilized ligand. Low surface concentration of ligand minimizes rebinding, avidity effects and favours reaction-limited adsorption but the resolution of the detector signal is the limiting factor. The resolution also limits determinations of b in the region lo-5 s.1 as only small amounts of analyte dissociate. The association equilibrium constant (K& or affinity constant, can be calculated as the ratio between association and dissociation rate constants. It can also be calcu lated from a series of experiments where the interaction reaches equilibrium during the sample injection [II**]. With an understanding of these artefacts that can interfere the BL4core measurements, relevant kinetic information can be obtained even from unpurified material in small quantities, which is not possible with, for example, fluorescence quench stop flow measurements.
283
284
Immunological
techniques
Examples of antibody-antigen
interactions
Recombinant
antibody fragments
AS an example of biospecific interaction analysis, four different monoclonal antibodies (mAbs) in tissue culture media and a monovalent purified antigen HIV-1 p24 were compared for alhnity and kinetic properties [ ll**]. By adsorption to the Fc region of aIlinky purified rabbit anti-mouse IgG immobilized on the sensor chip, each mAb could easily be characterized for antigen interaction and the sensor chip regenerated with 1OOmM hydrochloric acid. The qualitative information of relative affinity and kinetic properties was easily visualized by comparing sensorgrams run with the same concentration of antibody and antigen in an overlay plot. From calculation of kinetic and affinity constants, two of the antibodies were shown to have the same affinity but about a ftvefold difference in k, and lo. Affinities calculated from steady-state binding were less than f 50 per cent of values calculated from kinetic determinations.
The development of recombinant antibody technologies has been very rapid. The use of real-time biospecilic interaction analysis has been particularly useful for characterization of single chain (SC) Fv from primary libraries of rearranged V genes. The affinities, &, for these antibodies are in the range of 106-107 M-t, measured by fluorescence quench titration. A hapten specific human scFv was found to have a lo of 0.4 s-t [ 14**] and several other protein specific autoantibodies were found to have dissociation constants of around 10~2 s-r [ 15**]. It was suggested that these high relative rate constants with moderate affinity and high specificity for self antigens are generated during differentiation of B lymphocytes. Antibody fragments with such high dissociation rate constants are difftcult to analyze with enzyme-linked immunosorbent assay (ELISA) techniques as the half life of the complex is short. However, they can be used for reversible biosensors (M Malmqvist, unpublished data)
Another example is the determination of the kinetic constant of the Z-domain derived from protein A [I61 and five point mutated derivatives of it (L Cedergren, R An dersson, M Hartmanis, R Karlsson B Nilsson et al, abstract, page 18, Florence, August 1991, Application note 303,1992, Pharmacia, Biosensor). They were analyzed for their interaction with a fusion protein CD4-Fc, which was the immobilized ligand. With the assumption that all mutants were 100 per cent active, the results clearly show how the mutation L17D reduced k, IO-fold without any significant effect on lo, and mutation K35A increased the lo IO-fold without changing b. Such differences, which can be resolved by measuring kinetic properties, are of great importance for a more detailed understanding of biospecific interaction.
One strategy for building high aflinity antibodies in z&-o has been chain shuffling. High affinity hapten specific scFv antibodies with a K, of up to 0.9 x 109 M-l have been demonstrated by light and heavy chain shuffling [I4**]. The 300.fold increase in affinity over that of the original isolated scFv aphox- 15 from the naive library was found to be dominated by a loo-fold decrease in lo.
Kinetic analysis has also been performed for IgM and IgG anti-tetanus toxoid antibodies and Fab fragments [17*]. The regression analysis of the logarithmic decay of the dissociating complex fitted close to a straight line according to theory only for a Fab fragment with the tetanus toxoid antigen immobilized on the sensor chip. This clearly demonstrates artefacts that multiple binding sites can introduce. The same research group also analyzed the anti-hen lysozyme antibody 01.3, its Fab fragment, Fv fragment and Vtt domain [18*]. The determined affinities for Fab and Fv were surprisingly higher than those obtained in free solution by fluorescence quench titration and differed fivefold. This may be an effect of rebinding to unoccupied ligands or avidity effects from aggregated monomers of the antibody fragments as a relatively high surface concentration of ligand was used. Altschuh et al. [13*-l have made a detailed analysis of peptidemAb interaction due to single amino acid substitutions in a peptide from tobacco mosaic virus (TMV) protein. Three important amino acids were identified by analysis of kinetic characteristics of the mAb interaction and the paper also contains a discussion on bivalent binding of antibodies.
Epitope mapping Monoclonal antibodies are powerful tools for epitope mapping of antigens [ 191. Epitope mapping of HIV1 p24 core protein has been performed using BIAcore [2Oj. By a procedure based on pairwise analysis of 30 different monoclonal antibodies in tissue culture media, a two-dimensional surface like map was constructed. As the procedure is sequential, each step could be monitored, including the adsorption of antigen on the first mAb. Four different peptides were also identified that inhibit mAb binding without the use of labels on any component. Using the information obtained for different binding sites, Fagerstam et al. [20] studied sequential binding of different mAbs forming a hexamolecular complex by monitoring each step. This type of experiment will probably be important for a better understanding of function of multimolecular complexes. Ward et al. [21*] have mapped epitopes using antibodies against synthetic peptides of human interleukin (IL)-6. Epitopes on virus proteins have been mapped with conformational specific mAbs to cryptotopes, metatopes and neotopes on TMV protein both as two-site and multi-site binding assays [22*,23*]. Concentration
determinations
Concentration determinations in immunoassays are normally based on equilibrium measurements of immune complexes. In a flow system other parameters of interest are the dill&ion of substance to the sensor chip and the kinetic constants of the immobilized antibody. For
Surface piasmon resonance Malmqvist
further sensitivity and specificity a sandwich assay can be performed [24*,25*] as well as inhibition assays for haptens. A study was performed by VanCott et al. [2@*] on antibody reactivity to peptides from the envelope glycoprotein gpl6O of HIV-l. Concentration determinations for 25 HIV-1 positive serum samples tested on immobilized peptide obtained using ELISA (optical density) correlated well with values obtained using B&ore (RU). The sensitivity of BIAcore was determined by titration of a mAb against the ~3-100~ peptide RP135 to be 5 ng ml-l. In this report, BIAcore gave highly reproducible results and, under favourable conditions, similar sensitivity to ELISA. Using BIAcore specific components from analytical gel, filtration can be measured on-line. By analyzing the adsorption rate of scFv in the column effluent, the position for maximum concentration can be determined for identification of specific proteins in mixtures [15=-l. Conclusion
The development of a system based on SPR for labelfree detection of ligand-analyte interaction in real time has made kinetic/affinity information of antibody interactions accessible. The technology has in a short time been applied to several areas. A more general use of kinetic parameters will help us to describe biological systems and the instrument will also be of importance for epitope mapping, rapid identification of biospecific interactions and concentration determinations. Although the absolute value of kinetic constants based on one immobilized reactant to the flexible hydrophilic dextran matrix may differ systematically from values in free solution, the use of the system with knowledge of potential artefacts will rapidly give useful information of biospecific interactions. References
and recommended
Papers review, . ..
of particular interest, published have been highlighted as: of special interest of outstanding interest
1.
LIEDBERG
onance
JOHNSSON B, LOFTS S, LINDQVISTG: Immobilization of Proteins to a Carboxymethyldextran Modiied Gold Surface for Biospecfic Interaction Analysis in Surface PIasmon Resonance. Anal Biocbem 1‘991, 198:26%277. A carefully performed study of the important parameters for immobilization of proteins to the sensor chip in BIAcore. 5. ..
O’SHANNESSYDJ, BRIGHAM-BURKE M, PECKK: Immobilization Chemistries Suitable for Use in the BL4core Surface Plasmon Resonance Detector. Anal Biocbem 1992, 205:132-136. Functional groups such as aldehydes and thiols on ligands can be used for covalent immobilization to a sensor chip.
6. .
STENBERGE, PERSSONB, Roos H, URBANKZKYC: Quantitative Determination of Surface Concentration of Protein with Surface Plasmon Resonance by Using Radiolabeled Proteins. Colloid Interface Sci 1991, I43:513-526. A study of the correlation between the SPR signal response and surface concentration of protein. 7. .
8.
JONZDN
FAGERSTAM L,
the annual
period
of
10. .
KARISSON R, MICHAELSSON A, MATTSSONL: Kinetic Analysis of Monoclonal Antibody-Antigen Interactions with a New Biosensor Based Analytical System. J Immunol Methods 1991, 145:229-240. Kinetic and alfinity theory for the use of BIAcore and the kinetic characterization of unpurified mAbs. 11. ..
KARUSON R, ALTXHLJH D, VAN REGENMORTAL MHV: Measurement of Antibody Affinity. In Structure of Antigens, vol 1. Edited by Van Regenmortal MHV Boca Raton, Florida: CRC Press; 1992:127-148. The theory of kinetic and affinity measurements using BIAcore followed by examples of measurement. ALTSCHUH D, DUBS M-C, WEISS E, ZEDER-LETZ G, VAN REGENMORTAL MHV: Determination of Kinetic Constants for the Interaction between a Monoclonal Antibody and Peptides Using Surface Plasmon Resonance. BiocbemisDy 1992,
31:629%6304. IVARSSON B,
L~NDH
K,
IOF&
S,
PER~SONB, Rcxx H, RONNBERGI, SJOIANDERS, STENBERGE ET AL.: ReaI-time Biospectic Interaction Analysis Using Surface Plasmon Resonance and a Sensor Chip Technology. Biotedmiques 1991, 11:62@627. Presents an SPR based system, BIAcore, for real-time biospecific interaction analysis with some general applications. J~NS~ON U, MALMQWT M: Real Time BiospeciIic Interaction Analysis. The Integration of Surface Plasmon Resonance Detection, General Biospectic Interface and Microfluidics into one Analytical System. In Advances in Biosensors. Edited by Turner A. San Diego: JAI Press Ltd.; 1992:291-336. A general description of the development of biospecilic interaction anal~ ysis based on SPR over a 10 year peroid. 3. .
F
SJOIANDER S,
URBANKZKYC: Integrated Fluid Handling Systern for Biomolecular Interaction Analysis. Anal Chem 1991, 63:233%2345. An integrated fluidic system for biospecilic interaction analysis is described.
13. ..
. .
4.
Principles of Biosensing with an Extended Coupling Matrix and Surface Plasmon Resonance. Sensors Actuators 1993, in press.
BRIGHAM-BURKEM, EDWARDSJR, O’SHANNESSYDJ: Detection of Receptor-ligand Interaction Using Surface Plasmon Resonance: Model Studies Employing the HIV-l gp12OKD4 Interaction. Anal Biocbem 1992, 205:12%131. Description of operational characteristics of B&ore for the quantilication of biological macromolecules, i.e. stability under regeneration conditions, reproducibility and background adsorption from Streptomyces broth.
B, NYLANDERC, LUNDSTROM1: Surface Plasmon Resfor Gas Detection and Biosensing. Sensors Actuarors U,
LIEDBERG B, STENBERG E, LUNDSTROM I:
9. .
1983, 4:29%304. 2.
J Cbem Sot
12. .
reading
within
Efficient Covalent Immobilization of Ligands. Cbem &mm Issue 19’90, 21:15261528.
Lords S, JOHNSSON B: A Novel Hydrogel Matrix on Gold Surface in Surface Plasmon Resonance Sensors for Fast and
The authors discuss bivalent binding of mAbs to immobilized peptide ligands. They also describe the importance of using quantitative kinetic measurements, which allow a functional and dynamic analysis of an epitope in relation to structural characterization. 14. ..
MARKSJD,
GR~FFITHS AD,
WINTERG: Bypassing
Human
Antibodies
MALMQV~ST M, CLACKSON T, BYE JM, Immunisation: Building High Affinity by Chain Shuffling. Biotechnology 1992,
10:77%783. Describes a strategy for a&nity maturation of antibody fragments by chain shuffling and phage selection. The increase in affinity was dominated by a decrease in k+ 15. ..
GR~FFITHSAD, MALMQVIST M, MARKSJD, BYE JM, EMBLETONMJ, MCCAFFERTYJ, GORICK BD, HUGHES-JONESNC, HOOGE~OOM HR, WINTER G: Human Anti-self Antibodies with High Specticity from Phage Display Libraries. EMBO J 1993, 12:726734.
285
286
Immunological
techniques
Human autoantibodies from a naive library had characteristically very high association and dissociation rate constants. This paper also contains an example of on-line specific detection of proteins in gel filtration.
The interaction analysis of immobilized virus particles and mAbs showed that the virus retained conformational sensitive structures that usually partly denature on conventional solid phases.
16.
UH~N M, Gus B, NIISSON B, GATENBECK S, PHILIPSSONL, LINDBERGM: Complete Sequence of the Staphlococcal Gene Encoding Protein k J Biol Cbem 1984, 259:1695-1702.
17. .
MAIMBORG
DUBS M-C, ALTKHUH D, VAN REGENMORTALMHV Mapping of Viral Epitopes with Conformationally Specilic Monoclonal Antibodies Using Biosensor Technology. J Carom 1992, 597:391-396. Analysis of anti-neotope, anti-cryptotope and anti-metatope antibodies against TMV protein in a two-site binding assay.
A-C, MICHAEISSON A, OHUN M, JANSSON B, BORREBAECKCAK: Real Tie Analysis of Antibody-Antigen Reactive Kinetics. Scund J Immunol 1992, 35643-650. Analysis of antibodies of IgG, IgM and Fab for binding kinetics and affinity demonstrating effects of multiple binding sites. 18. .
BORREBAECKCAK, MALMBoRGA-C, FUREBRINGC, MICHAELSSON A, WARD S, OHUN M: Kinetic Analysis of Recombinant Antibody-Antigen Interactions: Relation between Structural Domains and Antigen Binding. Biotechnology 1992, 10:697-698. Comparison of antibody fragments for binding to immobilized antigen. 19.
VAN REGENMORTALMHV: Structural proaches to the Study of Protein Today 1989, 10:226272.
and Functional ApAntigenicity. ~mmunol
20.
FAGERXAMLG, FROSTFZU A, KARISSONR, LAR~~oNM, MALMQV~ST M, Burr H: Detection of Antigen-Antibody Interactions by J Mol Recogn 1990, 3:20%214. Surface PIasmon Resonance.
21. .
WARD IQ, SHI P-T, SIMPSON RJ: Binding of Anti-humaninterleukin-6 Monoclonal Antibodies to Synthetic Peptides of Human Interleukin-6 Studied Using Surface Plasmon Resonance. Biocbem Int 1992, 26~559-565. Epitope mapping of human IL-~ by peptide inhibition of antibody binds ing to antigen. 22. .
DUBS M-C, &ISCHUH D, VAN REGENMORTEI. MHV: Interaction between Viruses and MonoclonaI Antibodies Studied by Surface Plasmon Resonance. Immunol Lett 1991, 31:5’+64.
23. .
24. .
SOFAS S, MALMQV~ST M, RONNRERGI, STENBERGE: Bioanalysis with Surface Plasmon Resonance. Sensors Actuators 1991, 5:7%84. Determination of the concentration of IgE in serum using SPR and correlation with the I&%method. R, PERSIAN B, F;~GERSTAM LG, FROSTEU-IQFUSSONA, RONNBERGI: Biospeci6c Interaction Analysis Using Surface Plasmon Resonance Detection Applied to Kinetic, Biding Site and Concentration Analysis. J Chromatograph 1992, 597:397-410. Several examples of biospecific interaction analysis, including interaction analysis of immobilized luc repressor DNA and a protein fused to the lac repressor protein, concentration analysis and binding site analysis of insulin-like growth factor I and II and binding protein. 25. .
26. ..
VANCOTT TC, LOOMIS LD, REDFIELDRR, BIRX DL: Real-time Biospectic Interaction Analysis of Antibody Reactivity to Peptides from the Envelope Glycoprotein, gp160, of HIV1. J Imm Methods 1992, 1463163-176. A comparison of ELISA and BIAcore for concentration determinations of serum antibodies against gpl60 of HIV-l.
M Malmqvist, Pharmacia
Biosensor
AB, S-751 82 Uppsala, Sweden.
plasmon
measurement
resonance
for detection
of antibody-antigen
affinity
and
and kinetics
Magnus Malmqvist Pharmacia Biosensor
Kinetic and affinity information by analysis based
directly
of adsorption
analytical
AB, Uppsala, Sweden
on biospecific
of analyte
interaction
to immobilized
system. The analysis can be performed
using culture
supernatants.
Current
Opinion
valuable
1993, 5:282-286
lized ligand on the same spot and at the same time it occurs. The adsorption can be followed by SPR in realtime without labels and was first used for detection of antigen-antibody interaction by Liedberg et al. [ 11.
For the understanding of the structure-function relationship in an antibody-antigen interaction and for the technical use of antibodies, the characteristic properties of the interaction must be known. Accurate measurements of physical-chemical parameters, such as rate constants and affinity, are important as well as determination of binding epitopes. The practical use of antibodies for diagnostic purposes is well established, but new rapid techniques put higher demands on kinetic properties of the immunoreagents. Therapeutic use of antibodies also requires a more detailed understanding of the affinity and kinetics of antibodies, not least when human antibody fragments are to be used. The development of recombinant antibodies has provided tools to generate and modify properties of human antibody fragments. This development must be combined with rapid and reliable techniques to measure binding activity of generated molecules. Surface sensitive optical techniques, such as surface plasmon resonance (SPR), in combination with surface chemistry and accurate liquid handling, can be used for the rapid generation of kinetic and affinity information. Detection of the interactions without the use of label is applicable to any ligand-analyte pair and kinetic information of native molecules can be generated without purification of the components. This new technology has been particularly useful for detection and quantification of antigen-antibody interactions and is now also used for analysis of DNA protein and receptor-ligand pairs.
detection
a sensor
interactions.
in Immunology
Introduction
antibody-antigen
on
free of label and
It was found to be particularly
for analysis of antibody-antigen
SPR for the label-free
can be generated ligand
A system for flow of analyte samples over a surface with immobilized ligand and detection of the adsorbed analyte by SPR has been described by Jonsson et al. [2**,3*] The practical use of SPR was not possible until a system was commercially available under the name of BIAcoreR (Pharmacia Biosensor, Uppsala, Sweden) (Fig. 1). This system combines a detector, a sensor chip for immobilization of ligands and an integrated liquid handling fluidics for transport of samples and reagents to the place of interaction. The adsorption of anylate from the continuous flow of sample to the covalently immobilized ligand is measured as a function of time. After the sample pulse has passed the probed surface, the biospecific complex dissociates, measured as a decrease in response signal. The response [resonance signal (RU)] from the instru ment as a function of time is called a sensogram (Fig. 2). Most published work on kinetic and afhnity measurements based on surface sensitive techniques has been done using this instrument system. The interface between the liquid phase and the sensor chip has to fulfil several criteria regarding non-specific adsorption, immobilization procedures and stability. Sofas and Johnsson [4] have developed a surface consisting of a gold film covered by a layer of carboxylated dextran. Both large and small molecules can be covalently coupled to carboxyl groups on the sensor chip through amine, thiol and aldehyde functional groups [ 5**,6*I.
of
The matrix, which contains 2-3 per cent dextran is hydrophilic and coupled to the thin gold film through a linker layer without any crosslinking of the polymer [4]. This gives the matrix a flexible, hydrophilic, open structure with a high capacity for protein adsorption
interaction
The basic idea for any surface sensitive detector is to measure the adsorption of the analyte to an immobi-
Abbreviations ELISA-enzyme-linked K,association
equilibrium
immunosorbent constant;
w-single
282
assay; HIV-l-human
k,-association
chain; WI-surface
@
Current
rate constant;
immunodeficiency
kc
dissociation
plasmon
resonance;
TM-tobacco
Biology
Ltd ISSN 0952-7915
virus type 1; IL-interleukin;
rate constant; mosaic
mA&monoclonal virus.
antibody;
Surface
Light source
Photo detector array
dasmon
resonance
Malmavist
and affinity constants [ 11**,12*,13=]. The association rate constant (kJ can readily be obtained from a series of analyte concentrations. However, it is important to have an accurately determined concentration of active analyte for k, determinations. This can be done with BIAcore (R Karlsson, unpublished data) working under diflusion limited conditions and using a high surface concentration of ligand. The c&fusion rate to the surface is directly proportional to concentration so both diffusion and activity of the analyte are taken into consideration. For a k, of 106 M-1 s-1 the resolution of the instrument starts to be limiting as a very low concentration of analyte must be used.
Resonance signal [RUI
Fig 1. Real time SPR monitor. The figure shows the optical configuration together with the sensor chip and flow channels. The polarized light illuminates the sensor chip at the point where the biospecific interaction takes place and is reflected to the detector array. Through surface plasmon resonance, a dark spot can be seen on the detector for one angle of incidence. This angle for extinction of light is changed when analyte is adsorbed on the immobilized ligand on the sensor chip and is indicated on the figure as another dark line to the detector. [7*,8]. Both tissue culture media and bacterial broth give extremely low non-specific background on immobilized ligands [PI.
In order to define the liquid flow characteristics in the thin-layer channels and direct the liquid to the right place, a new type of miniaturized fluidic cartridge was created [lo-]. Regeneration conditions for the elution of all bound material have to be established and the activity of the immobilized ligand maintained. Most antibody systems need regeneration but there are exceptions. It was found that useful reagents for regeneration are strong acids, such as hydrochloric and phosphoric acid [p] The conditions have to be checked for every new immobilized l&and, analyte and regeneration agent. Kinetic and affinity
measurements
New techniques and methods for BIAcore as well as the interpretation of data are developing rapidly. More correlation data between other methods and techniques are needed as few results have been published. Results obtained from BIAcore should thus at present be regarded as estimates of alhnity and kinetic constants. The association phase of the so called sensorgram (Fig. 2) is a function of the kinetic properties of the reaction, as long as the mass transport of analyte to the immobilized ligand is not a limiting factor in the reaction. Flow rate, flow cell properties and diEusion coefficient determine the mass transport of analyte [lo*]. The law of mass action describing the reaction between analyte and l&and can be rewritten in terms of response signals derived from a flow system for calculation of kinetic
Time(s)
Fig 2. A sensorgram showing the response from the SPR detector [Resonance signal (RU)] when adsorption of an analyte to the immobilized ligand takes place. From the adsorption part of the sensorgram association rate constant can be determined. After the sample pulse has passed, dissociation of the complex on the surface begins, which can be used for calculation of dissociation rate constants.
The dissociation rate constant (b> can be determined from the association part of the sensorgram. However, in practice it is only valid for extremely high values. Sta tistical analysis based on non-linear regression analysis of the association part of a sensogram has the potential to give better resolution of kinetic properties. The b can be ascertained by measuring the dissociation of bound analyte in buffer flow after the sample pulse has passed the probed surface. However, it is important to ensure that analyte is free of aggregates to avoid mixed interactions [ 14**,15**], which can cause disturbances in the evaluation. Rebinding of analyte to unoccupied lig ands should also be avoided during the dissociation from immobilized ligand. Low surface concentration of ligand minimizes rebinding, avidity effects and favours reaction-limited adsorption but the resolution of the detector signal is the limiting factor. The resolution also limits determinations of b in the region lo-5 s.1 as only small amounts of analyte dissociate. The association equilibrium constant (K& or affinity constant, can be calculated as the ratio between association and dissociation rate constants. It can also be calcu lated from a series of experiments where the interaction reaches equilibrium during the sample injection [II**]. With an understanding of these artefacts that can interfere the BL4core measurements, relevant kinetic information can be obtained even from unpurified material in small quantities, which is not possible with, for example, fluorescence quench stop flow measurements.
283
284
Immunological
techniques
Examples of antibody-antigen
interactions
Recombinant
antibody fragments
AS an example of biospecific interaction analysis, four different monoclonal antibodies (mAbs) in tissue culture media and a monovalent purified antigen HIV-1 p24 were compared for alhnity and kinetic properties [ ll**]. By adsorption to the Fc region of aIlinky purified rabbit anti-mouse IgG immobilized on the sensor chip, each mAb could easily be characterized for antigen interaction and the sensor chip regenerated with 1OOmM hydrochloric acid. The qualitative information of relative affinity and kinetic properties was easily visualized by comparing sensorgrams run with the same concentration of antibody and antigen in an overlay plot. From calculation of kinetic and affinity constants, two of the antibodies were shown to have the same affinity but about a ftvefold difference in k, and lo. Affinities calculated from steady-state binding were less than f 50 per cent of values calculated from kinetic determinations.
The development of recombinant antibody technologies has been very rapid. The use of real-time biospecilic interaction analysis has been particularly useful for characterization of single chain (SC) Fv from primary libraries of rearranged V genes. The affinities, &, for these antibodies are in the range of 106-107 M-t, measured by fluorescence quench titration. A hapten specific human scFv was found to have a lo of 0.4 s-t [ 14**] and several other protein specific autoantibodies were found to have dissociation constants of around 10~2 s-r [ 15**]. It was suggested that these high relative rate constants with moderate affinity and high specificity for self antigens are generated during differentiation of B lymphocytes. Antibody fragments with such high dissociation rate constants are difftcult to analyze with enzyme-linked immunosorbent assay (ELISA) techniques as the half life of the complex is short. However, they can be used for reversible biosensors (M Malmqvist, unpublished data)
Another example is the determination of the kinetic constant of the Z-domain derived from protein A [I61 and five point mutated derivatives of it (L Cedergren, R An dersson, M Hartmanis, R Karlsson B Nilsson et al, abstract, page 18, Florence, August 1991, Application note 303,1992, Pharmacia, Biosensor). They were analyzed for their interaction with a fusion protein CD4-Fc, which was the immobilized ligand. With the assumption that all mutants were 100 per cent active, the results clearly show how the mutation L17D reduced k, IO-fold without any significant effect on lo, and mutation K35A increased the lo IO-fold without changing b. Such differences, which can be resolved by measuring kinetic properties, are of great importance for a more detailed understanding of biospecific interaction.
One strategy for building high aflinity antibodies in z&-o has been chain shuffling. High affinity hapten specific scFv antibodies with a K, of up to 0.9 x 109 M-l have been demonstrated by light and heavy chain shuffling [I4**]. The 300.fold increase in affinity over that of the original isolated scFv aphox- 15 from the naive library was found to be dominated by a loo-fold decrease in lo.
Kinetic analysis has also been performed for IgM and IgG anti-tetanus toxoid antibodies and Fab fragments [17*]. The regression analysis of the logarithmic decay of the dissociating complex fitted close to a straight line according to theory only for a Fab fragment with the tetanus toxoid antigen immobilized on the sensor chip. This clearly demonstrates artefacts that multiple binding sites can introduce. The same research group also analyzed the anti-hen lysozyme antibody 01.3, its Fab fragment, Fv fragment and Vtt domain [18*]. The determined affinities for Fab and Fv were surprisingly higher than those obtained in free solution by fluorescence quench titration and differed fivefold. This may be an effect of rebinding to unoccupied ligands or avidity effects from aggregated monomers of the antibody fragments as a relatively high surface concentration of ligand was used. Altschuh et al. [13*-l have made a detailed analysis of peptidemAb interaction due to single amino acid substitutions in a peptide from tobacco mosaic virus (TMV) protein. Three important amino acids were identified by analysis of kinetic characteristics of the mAb interaction and the paper also contains a discussion on bivalent binding of antibodies.
Epitope mapping Monoclonal antibodies are powerful tools for epitope mapping of antigens [ 191. Epitope mapping of HIV1 p24 core protein has been performed using BIAcore [2Oj. By a procedure based on pairwise analysis of 30 different monoclonal antibodies in tissue culture media, a two-dimensional surface like map was constructed. As the procedure is sequential, each step could be monitored, including the adsorption of antigen on the first mAb. Four different peptides were also identified that inhibit mAb binding without the use of labels on any component. Using the information obtained for different binding sites, Fagerstam et al. [20] studied sequential binding of different mAbs forming a hexamolecular complex by monitoring each step. This type of experiment will probably be important for a better understanding of function of multimolecular complexes. Ward et al. [21*] have mapped epitopes using antibodies against synthetic peptides of human interleukin (IL)-6. Epitopes on virus proteins have been mapped with conformational specific mAbs to cryptotopes, metatopes and neotopes on TMV protein both as two-site and multi-site binding assays [22*,23*]. Concentration
determinations
Concentration determinations in immunoassays are normally based on equilibrium measurements of immune complexes. In a flow system other parameters of interest are the dill&ion of substance to the sensor chip and the kinetic constants of the immobilized antibody. For
Surface piasmon resonance Malmqvist
further sensitivity and specificity a sandwich assay can be performed [24*,25*] as well as inhibition assays for haptens. A study was performed by VanCott et al. [2@*] on antibody reactivity to peptides from the envelope glycoprotein gpl6O of HIV-l. Concentration determinations for 25 HIV-1 positive serum samples tested on immobilized peptide obtained using ELISA (optical density) correlated well with values obtained using B&ore (RU). The sensitivity of BIAcore was determined by titration of a mAb against the ~3-100~ peptide RP135 to be 5 ng ml-l. In this report, BIAcore gave highly reproducible results and, under favourable conditions, similar sensitivity to ELISA. Using BIAcore specific components from analytical gel, filtration can be measured on-line. By analyzing the adsorption rate of scFv in the column effluent, the position for maximum concentration can be determined for identification of specific proteins in mixtures [15=-l. Conclusion
The development of a system based on SPR for labelfree detection of ligand-analyte interaction in real time has made kinetic/affinity information of antibody interactions accessible. The technology has in a short time been applied to several areas. A more general use of kinetic parameters will help us to describe biological systems and the instrument will also be of importance for epitope mapping, rapid identification of biospecific interactions and concentration determinations. Although the absolute value of kinetic constants based on one immobilized reactant to the flexible hydrophilic dextran matrix may differ systematically from values in free solution, the use of the system with knowledge of potential artefacts will rapidly give useful information of biospecific interactions. References
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1.
LIEDBERG
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JOHNSSON B, LOFTS S, LINDQVISTG: Immobilization of Proteins to a Carboxymethyldextran Modiied Gold Surface for Biospecfic Interaction Analysis in Surface PIasmon Resonance. Anal Biocbem 1‘991, 198:26%277. A carefully performed study of the important parameters for immobilization of proteins to the sensor chip in BIAcore. 5. ..
O’SHANNESSYDJ, BRIGHAM-BURKE M, PECKK: Immobilization Chemistries Suitable for Use in the BL4core Surface Plasmon Resonance Detector. Anal Biocbem 1992, 205:132-136. Functional groups such as aldehydes and thiols on ligands can be used for covalent immobilization to a sensor chip.
6. .
STENBERGE, PERSSONB, Roos H, URBANKZKYC: Quantitative Determination of Surface Concentration of Protein with Surface Plasmon Resonance by Using Radiolabeled Proteins. Colloid Interface Sci 1991, I43:513-526. A study of the correlation between the SPR signal response and surface concentration of protein. 7. .
8.
JONZDN
FAGERSTAM L,
the annual
period
of
10. .
KARISSON R, MICHAELSSON A, MATTSSONL: Kinetic Analysis of Monoclonal Antibody-Antigen Interactions with a New Biosensor Based Analytical System. J Immunol Methods 1991, 145:229-240. Kinetic and alfinity theory for the use of BIAcore and the kinetic characterization of unpurified mAbs. 11. ..
KARUSON R, ALTXHLJH D, VAN REGENMORTAL MHV: Measurement of Antibody Affinity. In Structure of Antigens, vol 1. Edited by Van Regenmortal MHV Boca Raton, Florida: CRC Press; 1992:127-148. The theory of kinetic and affinity measurements using BIAcore followed by examples of measurement. ALTSCHUH D, DUBS M-C, WEISS E, ZEDER-LETZ G, VAN REGENMORTAL MHV: Determination of Kinetic Constants for the Interaction between a Monoclonal Antibody and Peptides Using Surface Plasmon Resonance. BiocbemisDy 1992,
31:629%6304. IVARSSON B,
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PER~SONB, Rcxx H, RONNBERGI, SJOIANDERS, STENBERGE ET AL.: ReaI-time Biospectic Interaction Analysis Using Surface Plasmon Resonance and a Sensor Chip Technology. Biotedmiques 1991, 11:62@627. Presents an SPR based system, BIAcore, for real-time biospecific interaction analysis with some general applications. J~NS~ON U, MALMQWT M: Real Time BiospeciIic Interaction Analysis. The Integration of Surface Plasmon Resonance Detection, General Biospectic Interface and Microfluidics into one Analytical System. In Advances in Biosensors. Edited by Turner A. San Diego: JAI Press Ltd.; 1992:291-336. A general description of the development of biospecilic interaction anal~ ysis based on SPR over a 10 year peroid. 3. .
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SJOIANDER S,
URBANKZKYC: Integrated Fluid Handling Systern for Biomolecular Interaction Analysis. Anal Chem 1991, 63:233%2345. An integrated fluidic system for biospecilic interaction analysis is described.
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Principles of Biosensing with an Extended Coupling Matrix and Surface Plasmon Resonance. Sensors Actuators 1993, in press.
BRIGHAM-BURKEM, EDWARDSJR, O’SHANNESSYDJ: Detection of Receptor-ligand Interaction Using Surface Plasmon Resonance: Model Studies Employing the HIV-l gp12OKD4 Interaction. Anal Biocbem 1992, 205:12%131. Description of operational characteristics of B&ore for the quantilication of biological macromolecules, i.e. stability under regeneration conditions, reproducibility and background adsorption from Streptomyces broth.
B, NYLANDERC, LUNDSTROM1: Surface Plasmon Resfor Gas Detection and Biosensing. Sensors Actuarors U,
LIEDBERG B, STENBERG E, LUNDSTROM I:
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The authors discuss bivalent binding of mAbs to immobilized peptide ligands. They also describe the importance of using quantitative kinetic measurements, which allow a functional and dynamic analysis of an epitope in relation to structural characterization. 14. ..
MARKSJD,
GR~FFITHS AD,
WINTERG: Bypassing
Human
Antibodies
MALMQV~ST M, CLACKSON T, BYE JM, Immunisation: Building High Affinity by Chain Shuffling. Biotechnology 1992,
10:77%783. Describes a strategy for a&nity maturation of antibody fragments by chain shuffling and phage selection. The increase in affinity was dominated by a decrease in k+ 15. ..
GR~FFITHSAD, MALMQVIST M, MARKSJD, BYE JM, EMBLETONMJ, MCCAFFERTYJ, GORICK BD, HUGHES-JONESNC, HOOGE~OOM HR, WINTER G: Human Anti-self Antibodies with High Specticity from Phage Display Libraries. EMBO J 1993, 12:726734.
285
286
Immunological
techniques
Human autoantibodies from a naive library had characteristically very high association and dissociation rate constants. This paper also contains an example of on-line specific detection of proteins in gel filtration.
The interaction analysis of immobilized virus particles and mAbs showed that the virus retained conformational sensitive structures that usually partly denature on conventional solid phases.
16.
UH~N M, Gus B, NIISSON B, GATENBECK S, PHILIPSSONL, LINDBERGM: Complete Sequence of the Staphlococcal Gene Encoding Protein k J Biol Cbem 1984, 259:1695-1702.
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A-C, MICHAEISSON A, OHUN M, JANSSON B, BORREBAECKCAK: Real Tie Analysis of Antibody-Antigen Reactive Kinetics. Scund J Immunol 1992, 35643-650. Analysis of antibodies of IgG, IgM and Fab for binding kinetics and affinity demonstrating effects of multiple binding sites. 18. .
BORREBAECKCAK, MALMBoRGA-C, FUREBRINGC, MICHAELSSON A, WARD S, OHUN M: Kinetic Analysis of Recombinant Antibody-Antigen Interactions: Relation between Structural Domains and Antigen Binding. Biotechnology 1992, 10:697-698. Comparison of antibody fragments for binding to immobilized antigen. 19.
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FAGERXAMLG, FROSTFZU A, KARISSONR, LAR~~oNM, MALMQV~ST M, Burr H: Detection of Antigen-Antibody Interactions by J Mol Recogn 1990, 3:20%214. Surface PIasmon Resonance.
21. .
WARD IQ, SHI P-T, SIMPSON RJ: Binding of Anti-humaninterleukin-6 Monoclonal Antibodies to Synthetic Peptides of Human Interleukin-6 Studied Using Surface Plasmon Resonance. Biocbem Int 1992, 26~559-565. Epitope mapping of human IL-~ by peptide inhibition of antibody binds ing to antigen. 22. .
DUBS M-C, &ISCHUH D, VAN REGENMORTEI. MHV: Interaction between Viruses and MonoclonaI Antibodies Studied by Surface Plasmon Resonance. Immunol Lett 1991, 31:5’+64.
23. .
24. .
SOFAS S, MALMQV~ST M, RONNRERGI, STENBERGE: Bioanalysis with Surface Plasmon Resonance. Sensors Actuators 1991, 5:7%84. Determination of the concentration of IgE in serum using SPR and correlation with the I&%method. R, PERSIAN B, F;~GERSTAM LG, FROSTEU-IQFUSSONA, RONNBERGI: Biospeci6c Interaction Analysis Using Surface Plasmon Resonance Detection Applied to Kinetic, Biding Site and Concentration Analysis. J Chromatograph 1992, 597:397-410. Several examples of biospecific interaction analysis, including interaction analysis of immobilized luc repressor DNA and a protein fused to the lac repressor protein, concentration analysis and binding site analysis of insulin-like growth factor I and II and binding protein. 25. .
26. ..
VANCOTT TC, LOOMIS LD, REDFIELDRR, BIRX DL: Real-time Biospectic Interaction Analysis of Antibody Reactivity to Peptides from the Envelope Glycoprotein, gp160, of HIV1. J Imm Methods 1992, 1463163-176. A comparison of ELISA and BIAcore for concentration determinations of serum antibodies against gpl60 of HIV-l.
M Malmqvist, Pharmacia
Biosensor
AB, S-751 82 Uppsala, Sweden.