WO2001079848A1

WO2001079848A1 - Method for determination of binding with natural receptors

Info

Publication number: WO2001079848A1
Application number: PCT/NL2001/000265
Authority: WO
Inventors: Abraham Van Der Gaag; Edwin Cornelis Albert Stigter; Stephan W illem Frederik Marie VAN HÖVELL TOT WESTERFLIER; Elwin Robbert Verheij; Marco Gaspari
Original assignee: Nederlandse Organisatie voor Toegepast Natuurwetenschappelijk Onderzoek TNO
Current assignee: Nederlandse Organisatie voor Toegepast Natuurwetenschappelijk Onderzoek TNO
Priority date: 2000-03-31
Filing date: 2001-03-30
Publication date: 2001-10-25
Anticipated expiration: 2002-09-30
Also published as: AU2001246943A1

Abstract

A method is described for the reversible linking of a natural receptor to a surface, which receptor is present in cell membrane fragments, where the receptor is joined to a coupling substance such as biotin, which can enter into a specific and reversible bond with an antibody, such as anti-biotin, and the antibody is joined to the sensor surface. Specific binding between the receptor and a ligand can be determined by bringing the sensor coated with the receptor into contact with the ligand and measuring the interation between the receptor and the ligand, for example with Mach-Zehnder interferometry.

Description

Method for determination of binding with natural receptors

Field of the invention

The invention is in the field of the development, using biosensors, of new medicinal products which exhibit binding with natural receptors, and in the field of further study of binding to membrane-bound antigens or transport channels.

Background to the invention

There is a continuing need for new medicinal products, especially medicinal products which can enter into a controllable and specific interaction with receptors in the body. This applies particularly to receptors bound to cell membrane, such as receptors which form part of the central nervous system. For this development it should be possible to test as large a number as possible of candidate medicinal products in vitro for possible interaction with such receptors.

Optical measurement techniques are suitable for measuring the interactions which have been described. By the binding of ligands to receptors there will be a change in the refractive index close to the sensor surface.

One of the detection methods to be used in such tests involves Surface Plasmon Resonance (SPR), more particularly the BIACORE® (Pharmacia). The BIACORE is an integrated whole consisting of an optical detection unit, a flow-through system and a sensor surface. This surface is a replaceable sensor chip consisting of a glass slide with a thin gold layer to which a coating consisting of carboxylated dextran is applied. A receptor molecule can be linked to this coating (Lδfas & Johnsson 1990).

Detection in this system is based on surface plasmon resonance (SPR), which is essentially the measurement of changes in the refractive index which are caused by binding of molecules to the sensor surface. At a particular wavelength and angle of incidence, light energy is absorbed by free electrons in the thin metal film and the intensity of light reflected will consequently decrease. The angle at which this phenomenon occurs changes with the mass on the sensor surface. The final measured difference in refractive index is dependent on the rate of association and dissociation of a receptor molecule and the relevant ligand.

With SPR, more specifically the BIACORE 2000 and 3000 series, it is possible to observe a change in refractive index as a result of binding to receptor molecules of particles with a mass of a few hundred daltons. The quantity of particles to be bound, and thus the concentration thereof, must then be very high, however. This means that the practical applicability of SPR for the activities described is limited.

The changes in refractive index as described above are in the order of magnitude of 10^"7 to 10^"8. With a sensor based on Mach-Zehnder Interferornetry (MZI) it is possible to observe these small changes in refractive index (R. Heideman, 1993). Changes in refractive index are also measured with MZI. The detection is not based on measurement of changes in the intensity of light, as in the case of SPR, but on measurement of phase differences of the light. Because the change in phase of light can be measured more accurately than changes in the intensity of light, the sensor based on MZI is better able to accurately measure changes in refractive index due to low- molecular-weight components.

The principle of specific biological interactions is widely used in analytical chemistry. Good examples of this include immunoassays, receptor assays and immuno-affinity for sample preparation. In combination with mass spectrometry, the use of immuno-affinity in particular is of interest for the isolation of very small quantities of the compound(s) to be measured from complex samples (blood, urine, tissue, etc.). Immuno-affinity sample preparation is used either separately or on-line. An example of the latter is the use of immuno-affinity columns in combination with coupled liquid chromatography-mass spectrometry (LC-MS). In addition to LC-MS, MALDI-ToF-MS is a widely used mass spectrometry technique which offers interesting possibilities for the application of biospecific interactions.

MALDI-ToF-MS stands for 'Matrix Assisted Laser Desorption/Ionization Time- of-Flight Mass Spectrometry'. This technique is mainly used for analysis of biomacromolecules such as peptides, proteins, DNA, RNA, polysaccharides, etc.

The principle of operation is as follows. The sample, either one compound or a mixture of several compounds, incorporated into a suitable solvent, is put onto a 'target'. The target consists of a conductive material, usually stainless steel, but other materials, which may be given special coatings (e.g. gold), are also used. A 'matrix' is also used. This is added before, at the same time as or after the sample is applied. During evaporation of the solvent the matrix will crystallize out and the compounds to be measured will be incorporated into the crystal structure. The target with the prepared sample(s) on it is then introduced into the MALDI-MS. A laser is fired at the prepared sample and the compounds are thus desorbed and ionized. Pulsed UN lasers are usually employed. IR lasers are also used. The matrix plays a significant role in this process. The choice of matrix depends on the type of compound, the method of preparation of the sample/matrix mixture, the wavelength of the laser, and other factors. When UN lasers are used the matrix is a strong organic acid which has the property of absorbing UV radiation owing to the presence of an aromatic structure element, such as 2,5-dihydroxybenzoic acid, α-cyanocinnamic acid, sinapic acid, etc. Other matrix compounds are used for IR lasers.

The ions formed are then measured with a ToF mass spectrometer. The MALDI process results mainly in protonated molecules [M+H]⁺ and deprotonated molecules [M-H]^". Depending on the mass spectrometer settings, either the positive or the negative ions are measured. In this way it is possible to determine the molecular mass of a compound (or mixture of compounds). Structural data can be obtained by fragmentation of the ions by PSD, MS/MS or MS".

MALDI in combination with ToF-MS and pulsed UN lasers is at the moment the most widely used form of the technique. Other lasers and/or other types of mass spectrometers can also be used, however. The MALDI technique also has similarities to other desorption techniques such as FAB (Fast Atom Bombardment), SIMS (Secondary Ion Mass Spectrometry) and PD (Plasma Desorption). In these techniques xenon atoms, caesium ions and nuclear fission products (californium-252), respectively, are used instead of radiation to desorb and ionize the compounds.

In addition to MALDI there is a virtually identical technique, called SELDI (Surface Enhanced Laser Desorption/Ionization) [3]. The fundamental similarities between all these desorption/ionization techniques give reason to assume that this invention can also be used with these techniques.

With regard to biospecific interactions, MALDI-ToF-MS offers the interesting possibility of immobilizing molecules with a specific affinity for other molecules on the MALDI target. With such a bioaffinity target the compounds to be measured can be captured selectively from complex mixtures and measured by MALDI after removal of non-binding material. This is described in the scientific literature for antibodies and a number of other biospecific interactions [1, 2, 3]. No published material has been found with regard to the immobilization of receptors, and in particular membrane- bound receptors. Description of the invention

The invention relates to a method for determining specific binding between a natural receptor and its ligand, by means of the reversible immobilization, on sensor surfaces, of receptors, proteins or other biological molecules bound to cell membranes or to membranes of subcellular structures (organelles). The methods according to the invention are defined in the appended claims. A "natural receptor" or a "specific binding substance" here means a biological molecule which has a specific binding partner, usually a molecule which is present on a cell membrane or a subcellular membrane, such as an organelle. It may be a receptor in the narrower sense, but also another specific binding molecule, such as an antigen, a transport protein, etc. This specific binding partner is referred to as "ligand". The expression "present in cell membrane fragments" means present in such a way - usually on cellular material - that the natural functionality is retained.

The immobilization takes place via a coupling substance to which the receptor is joined and a substance binding or complexing therewith, referred to as antibody, which is joined to the sensor surface. The coupling substance can be an antigen, for example, and the antibody is then an antibody to the antigen. Examples are biotin and anti- biotin, digoxigenin and anti-digoxigenin. It is a prerequisite that the interaction between the antigen and antibody can be regenerated.

In the literature there are reports of the immobilization of membrane-bound biological molecules by first isolating these molecules in a purified form from their natural environment. The molecules are then immobilized on the surface of a synthetic membrane. The big disadvantage of this immobilization technique is that the biological molecules must first be isolated in a purified form. During the purification there is usually a change in the structure of the biological molecule and thus also the biological activity and specificity. After the purification the biological molecules are placed in a synthetic membrane. The composition of this membrane differs from the natural membrane from which the biological molecule originates. This can have the effect that once again there is a change in the biological activity and specificity. When measurements are made with coatings of this kind, it is thus questionable whether what is measured really is what one wants to measure. It is therefore doubly important to immobilize biological molecules of this kind without taking the molecules out of their natural environment. A second problem which occurs is that the interaction between the ligand and the relevant receptor, for example, can no longer be broken. This means that the sensor surface can no longer be regenerated. This problem arises because a cascade mechanism is usually present at cellular level. An example of this is the mechanism of the receptor bound to G protein. At the moment when a ligand binds to the receptor the α-subunit will be detached from the G protein complex. This α-subunit is responsible for energy transfer to the membrane-bound enzyme adenylate cyclase. In a closed system such as the cell the α-subunit is circulated. After the α-subunit has transferred the energy to the enzyme it can bind to the G protein again. At that moment the ligand can be detached again from the receptor molecule. This principle of circulation does not take place on a sensor surface. As a result, a sensor surface can only be used once.

The solution to this problem is to immobilize the membranes in a generic and reversible way. This means that each membrane type can be bound to the sensor surface in the same way. When the interaction between the receptor and the ligand has taken place, the whole complex of membranes, with receptors, and the bound ligand is removed from the sensor surface. After this, membrane fragments can be bound to the sensor surface again, after which the interaction between the receptor and a new ligand to be tested can be measured.

Membranes can be bound generically and reversibly in the ways described below.

The antibody is covalently bound to the sensor surface. These antibodies recognize a molecule of low molecular weight. Examples are biotin or substances with a comparable molecular mass. The bond between the sensor surface and the antibody can be a covalent bond. The sensor surface can be activated by plasma polymerization, for example, in such a way that the surface contains functional groups. A usable functionalization is that with ethylenediamine. Further functionalization can take place with thiolane, for example, with the formation of a thiol, to which PMPI (p-maleidophenyl isocyanate), for example, can bind. PMPI forms an isourea bond (-NH-CO-NH-) with an a ine or a carbamate bond (-NH-CO-O-) with, for example, a saccharide of the antibody. When the sensor surface is functionalized with another reactive group, other conventional homo- or hetero-bifunctional crosslinkers can be used (see, for example, Wong, 1991). Examples of crosslinkers of this kind are disuccinimidyl suberate (DSS), dimethyl pimelimidate (DMP), m-maleimidobenzoyl sulphosuccinimide ester (Sulpho-MBS), N-succinimidyl-4-azido-salicylate (NHS- ASA), and l-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC). The biotin or other coupling substance is covalently bound to a lectin or a substance which, just like lectin, can enter into an interaction with a membrane, for example via the saccharide components thereof. Other interactions between membrane components and receptor molecules are possible. Examples of membrane components include cholesterol, Na/K pumps, membrane-bound enzymes and proteins, glycoproteins and glycolipids. A labelled lectin has now been made which can be bound to the sensor surface by means of the antibodies to biotin. A solution of membrane fragments is then passed over the sensor surface. The membrane fragments will now bind to the sensor surface as a result of the fact that the lectin enters into an interaction with the membrane. This interaction is in most cases not reversible. If it is, the lectin can be covalently bound directly to the sensor surface. The membranes can be removed from the sensor surface by breaking the bond between the covalently bound antibody and the labelled lectin. The labelled lectin can then be offered again, after which membrane fragments can bind to the sensor surface again.

In this way 1.3*10¹⁰ to 2.7*10¹⁰ receptors per mm² of sensor surface can be linked, depending on the receptor density in the membrane. In comparison with the literature (Kalb, 1992 and Cooper, 2000), where a 2% surface density of the receptors is attained, in this case a surface density of 68% is achieved.

As a variant of what has been described above, the membrane fragments can be directly biotinylated. In most cases this causes a deterioration in the result, because the biotin will also bind to the receptor molecules, as a result of which the receptor molecules can no longer recognize and bind their ligand. In addition, use can also be made of the immobilization of other receptor molecules (such as antibodies, receptors, DNA, etc.) in order to bind the membrane fragments. The receptor molecules then recognize biomolecules in the membrane. Examples include antibodies to lipopolysaccharides and cholesterol.

A linked, used sensor can be regenerated by incubation or injection with a regeneration liquid. Depending on the coupling substance and the antibody, this can be an acid, a base, a salt (high concentration), a soap or an organic solvent, or a combination of these. For biotin-antibiotin a sodium hydroxide solution of 25 mM, for example, is satisfactory. In other cases a different solution may be needed for regeneration of the sensor surface (Tsang, 1991).

The surface on which a ligand can be immobilized can also be a MALDI target, for example, instead of a sensor surface. The invention is consequently applicable to interactions which are not reversible, e.g. receptor-ligand interaction with a G protein/receptor mechanism. The physicochemical processes during MALDI lead to the breaking of this interaction. It is thus possible to detect the ligand.

The invention makes it possible also to apply detection by MALDI-ToF-MS, in addition to the stated detection techniques (SPR and MZI), so that, in addition to the quantification of an interaction, e.g. receptor-ligand binding, it is also possible to identify the ligand (molecular mass and fragmentation by PSD, MS/MS or MS").

The major advantage of this approach is that only the compounds with biological activity (agonistic or antagonistic) are detected. Antibodies recognize molecules only on the basis of particular structural elements and there is no universally applicable relationship between immuno-affinity and biological activity (receptor affinity).

The possible applications include:

1) Identification of ligands in complex mixtures, e.g. combinatorial libraries, natural products (extracts from plants, micro-organisms, animals, organs, etc.), active metabolites of medicinal products, active contaminants in medicinal products, active decomposition products of medicinal products, etc. The presence of one or more active compounds (e.g. receptor ligands) is easy to measure by classical techniques (receptor assays, biological assays, etc.), but the identification of the active components can be very time-consuming (extensive clean-up processes such as fractionation, followed by activity measurements on the fractions and identification). This invention greatly simplifies this process, because not only is the interaction of a ligand with a receptor detected but identification of the ligand is also possible in the same experiment.

2) Selective clean-up technique for quantitative mass spectrometry determination methods for the bioanalysis of medicinal products and body substances in biological matrices. The compound is isolated from a complex mixture by a specific interaction. Non-binding components in the sample are quantitatively removed. Since a mass spectrometer separates compounds on the basis of molecular mass, internal standards can also be used here (similar compound with a different mass from the compound which is to be determined) in order to improve the accuracy of the measurement. Example 1

1. Introduction

Here the development is described of a N2 receptor coating with a removable receptor-ligand layer for application to a Mach-Zehnder Interferometer (MZI). A ligand (agonist) of the N2 receptor is vasopressin, in particular arginine-vasopressin (ANP). Experiments with radioreceptor analysis (RRA) as well as experiments made with a biosensor on the basis of 'surface plasmon resonance' (SPR) have shown that it is possible to immobilize receptor membrane fragments (RMF's) generically and in a regenerable way on the MZI sensor surface.

It is here described how a bio-interface (RMF's) must be implemented on an integrated Mach-Zehnder Interferometer (IMZI). The IMZI chips with the receptor biocoating obtained must meet the sensor specifications with regard to:

• selective detection of the AVP agonist of the N2 receptor

• regenerability of the sensor surface

• a generic detection system for testing different types of membrane-bound receptors.

2. Material and methods

The IMZI sensor chips, purchased from Mierij Meteo bv, were given a N2- RMF biocoating. Two techniques were used for the immobilization of the N2-RMF. These coatings consisted of:

1) biotinylated N2-RMF bound to the anti-biotin, which in turn was covalently bound to the plasma-polymerized sensor surface, and

2) natural N2-RMF bound to the anti-biotin on the sensor surface via a generic intermediary molecule, namely lectin-biotin, where lectin binds to the saccharide components of the RMF's and/or is incorporated into the lipid structure of the membranes.

2.1. Preparation of the V₂ receptor membrane fragments (RMF)

The N2 receptor cells were obtained by cloning and isolating the cell line of the type 2936hN2R. By treating the resultant cell suspensions with lysis buffer, N₂ receptor membrane fragments (RMF) were obtained in accordance with the protocol described in Appendix B to the Progress Report phase 2. Cell growth:

L-15 medium (Liebowitz's medium; Gibco 41300-021) was used for growth in suspension (in spinner flasks), supplemented with 10% FBS, 2mM glutamine, pen/strep, 0.1% Pluronic F-68 (Gibco 24040-032), 250 μg/ml hygromycin (Gibco).

The maximum cell density is 10⁶ cells/ml. The cells are thinned out twice a week.

Membrane preparation:

Membrane buffer 100 mM TRIS-HCl, pH 7.5 (room temperature), 10 mM MgCl₂

Lysis buffer 50 mM TRIS-HCl, pH 7.5 (room temp.), 3 mM EDTA, 2 mM MgCl₂

1. The cells are collected and centrifuged at 700 rpm for 5 minutes at room temperature.

2. The cells are resuspended in ice-cold lysis buffer and homogenized in a glass-glass homogenizer with 12-15 movements.

3. The membranes are centrifuged at 21.000 g for 30 minutes at 4°C.

4. The pellet is finally resuspended in ice-cold membrane buffer and kept at -80°C.

2.2. Functionalizing the surface of the IMZI chip (SiOj)

The IMZI chips were cleaned with the aid of a microwave-based plasma etching apparatus in an argon/oxygen atmosphere for 5 minutes at a 100% microwave output of 1250 W. Immediately afterwards, without interference, the samples were exposed to an ethylenediamine vapour plasma in the same microwave chamber for 30 seconds, at 5% microwave output. The gas flow rate of 15 ml/min was controlled by a mass flow control system. By means of plasma polymerization of MZI chips in ethylenediamine, the surfaces were functionalized with amino groups which could be used for the further linking of molecules to the surface, resulting in 'Optical qualified Functionalized Surfaces' (OFS). The amino groups on the chips polymerized with plasma are modified to thiol groups (-SH) by means of thiolanization with 1 mg/ml thiolane in a borate buffer solution.

An anti-biotin antibody was linked to the surface of the IMZI chip by incubating the chip in 1 mg/ml N-[p-maleimidophenyl]isocyanate (PMPI) in borate buffer for 1 hour and then in 50 μg/ml anti-biotin in borate buffer for 1 hour.

The sensor surface can be coated with different functional groups. Examples include carboxyl, amino, thiol and epoxide groups. With the aid of homo- or hetero-bifunctional crosslinkers, the receptor molecules to which the membrane fragments bind can be bound to the sensor surface. 2.3. Biotinylation of RMF 's

RMF derivatized with PMPI (biotinylation)

1 ml 0.5 mg/ml NHS-LC biotin was added to 1 ml 0.5 mg/ml 1-cysteine (both in 50 mM borate buffer, pH 8.9) and the reaction mixture was stirred for 2 hours at room temperature. 0.8 mg PMPI in 50 μl DMSO was added to this mixture and the mixture thus obtained was then stirred for 1 hour at room temperature in darkness.

2 ml RMF solution (2 mg/ml protein, membrane buffer) were then added, and the resultant mixture was stirred for at least 2 hours in darkness and then stored at 4°C until it was used.

2.4. Biotinylated Concanavalin A

50 μl 0.3 mg/ml NHS-LC biotin in 50 mM borate buffer (pH 8.9) and 850 μl 50 mM borate buffer (pH 8.9) were added to 100 μl Concanavalin A, and the mixture was stirred for 2 hours and then stored at 4°C until it was used.

2.5. Immobilization of RMF's on the sensor surface

The RMF's were immobilized on the anti-biotin IMZI chips in two different regenerable ways:

• in one step: before binding of biotinylated RMF, or

• in two steps: by binding of biotin-lectin and unmodified RMF, respectively.

2.6. IMZI experiments

The double cuvette was mounted on the optical windows of the IMZI.

The first specific receptor experiments on the IMZI setup were carried out with an IMZI chip coated with a mouse anti-biotin layer prepared as described in 2.2.

The sample - which can be a dilute solution of biotinylated RMF's, unbiotinylated RMF's or lectin-biotin in running buffer (Hepes-buffered NaCl, HBS) for immobilization of the RMF's or a solution of AVP in HBS for the detection of ANP - was injected into one or both cuvettes and mixed by rinsing again with a pipette valve. The washing steps were carried out with HBS. 2.7. Calculation of the A VP sensitivity from the IMZI phase shift response

The principle of measurement is based on the change in the refractive index in the evanescent field, measured with an interferometer. For a flat optical wave as sensing element the literature (R. Heideman, 1993) discusses a theoretical relationship between the phase shift and the effective change in the refractive index of the mode, the duration of the interaction, the wavelength of the laser light used and the change in the refractive index of the covering, including reactions on the surface.

For a homogeneous protein layer on the surface of the IMZI sensor, with a refractive index of 1.45 for protein and 1.33 for water, the change in the refractive index can be calculated from the phase shift. The refractive index increases by 0.001 on absorption of 1 ng/mm² protein on the sensor surface (Pharmacia Biosensor AB, 1990).

This results in a relationship between the phase shift and the protein concentration on the surface Am, in pg/mm :

ΔΦ = 2.7 10³ * An * 2π and Δm = 370 * ΔΦ(2π) pg/rnrn²

A phase shift for IMZI of 0.012π corresponds to a protein density on the surface of 3.7 pg/rnrn².

3. Results and discussion

The aim of the experiments was to implement the biocoating of vasopressin receptor-membrane fragments (N2-RMF) on the integrated Mach-Zehnder Interferometer (IMZI). The aim was to be able to show that

• IMZI can be used for selective detection of the N2 agonist ANP, and

• the sensor surface can be regenerated.

IMZI chips with a biocoating which contained another receptor bound to the membrane, namely the β-adrenoceptor, were also prepared and tested for sensitivity to ANP in order to investigate whether:

• the sensor can be used as a generic detection system for different types of receptors bound to a membrane. 3.1. Vasopressin-RMF biocoating on the IMZI and detection of A VP

The IMZI sensor chips were given a N2-RMF biocoating as described under Material and methods. These coatings consisted of:

• biotinylated N2-RMF bound to the sensor surface modified with anti-biotin, and

• natural N2-RMF bound to the anti-biotin on the sensor surface via a generic intermediary molecule, lectin-biotin, where lectin binds to saccharide components of the RMF's and/or is incorporated into the lipid structure of the membranes.

Figure 1 shows the on-line immobilization of biotinylated N2-RMF on an IMZI chip with anti-biotin coating. The figure shows a time plot of the IMZI response expressed in response units of a phase shift of 2π (RU) during immobilization. A phase shift of 0.4 RU was obtained after injection of 50 μl 25 μg/ml biotinylated N2-RMF. A phase shift of about 0.3 RU was left after washing with running buffer. This indicates that a N2-RMF-biotin-»-antibiotin chip has actually been produced.

Figure 2 shows a response time plot during the binding of ANP to the N2-RMF- biotin-»-antibiotin chip. Two minutes after injection of 500 μM ANP the response time curve was broken off because an air bubble had got onto the sensor surface during the ANP injection. After stabilization of the signal, a phase shift of 0.055 RU was achieved. Taking into account the relationship between phase shift and protein concentration on the surface, as described in Material and methods, this corresponds to a protein density on the surface of about 18 pg/mm². Replacement of the solution by 750 μM ANP increased the IMZI response to 0.12 RU, corresponding to a density of bound ANP on the surface of 45 pg/mm .

The fact that the response did not increase further with a higher ANP concentration, of 875 μM, shows that the saturation level for binding had been attained.

Figure 3 shows a response time plot for immobilization of unbiotinylated (natural) RMF via biotinylated lectin (Concanavalin A, ConA) on the IMZI chip with antibiotin coating. The immobilization steps of lectin-biotin and N2-RMF's, respectively, are reproduced in the IMZI response curve. Injection of ANP resulted in a phase shift of about 0.06 RU, which corresponds to 22 pg/mm² bound ANP on the sensor surface. 3.2. Regeneration

Between the various experiments the chip was washed with 25 mM NaOH. This regeneration step led to removal of all the biological material which was deposited on the antibiotin layer. This indicates that both immobilization methods - biotinylated RMF and lectin-biotin with unbiotinylated RMF - are regenerated to the level of antibiotin«-biotin.

3.3. β-Adrenoceptor-RMF biocoating on the IMZI and a negative control

A negative control was performed with an RMF biocoating which contained an alternative receptor bound to the membrane, namely the β-adrenoceptor.

Figure 4 shows a response time curve which results from the IMZI experiments with a chip surface coated with antibiotin. The binding of lectin-biotin results in a residual phase shift of 9 RU after washing with membrane buffer. A phase shift of about 4 RU followed afterwards on injection of β-adrenoceptor RMF.

This shows that an alternative receptor bound to the membrane can also be effectively immobilized. The method in which biotinylated lectin (ConA) is used is found to be a generic immobilization method.

The subsequent injection of ANP on the β-adrenoceptor-lectin-biotin-»- antibiotin chip thus obtained resulted in an initial phase shift of 0.2 RU. After washing with buffer solution, however, the response signal returned to the base value.

Injection of ANP did not yield any residual IMZI response. This shows that the binding of ANP to the biocoating of the N2 receptor on the IMZI chip is specific.

Conclusion

The results obtained with RRA and SPR could be reproduced by experiments with the integrated Mach-Zehnder Interferometer (IMZI). All the aims - specific detection of ANP, the regeneration of the sensor chip and generic immobilization of receptors bound to the membrane - were achieved.

4. References:

• Cooper, M.A., Hansson, A., Lδfas., and Williams, D.H., A vesicle capture sensor chip for kinetic analysis of interactions with membrane-bound receptors,

Analytical Biochemistry 277, 196-200, 2000. • Heideman, R.G., "Optical waveguide based evanescent field immunosensors", Thesis, ISBN 9005679 3, 1993, University of Twente.

• Kalb, E. et al., Biochim. Biophys. Acta, 1992, 1103, 307-316.

• Pharmacia Biosensor AB, Lδfas en Johnsson, "BIAcore™ Methods Manual" November 1990 edition, pp. 3-4.

• Tsang, N.C.W. and Wilkins, P.P., Optimum dissociating conditions for irnmuno affinity and preferential isolation of antibodies with high specific activity, Journal of Immunological Methods, 138, 1991, 291-299.

• Wong, S.S., Chemistry of Protein Conjugation and Crosslinking, CRC Press, ISBN 0-8493-5886-8, 1991.

Example 2

1. Introduction

As an extension to the other examples, the mass spectrometer detection of arginine-vasopressin (ANP) after binding to the N2 receptor immobilized on the MALDI target is described here. In order to prove that the invention works it was important to obtain answers to the following questions:

1) Is it possible to break the receptor-ligand interaction with MALDI (UN laser)?

2) How strong are the signals from the membrane components and the chemicals of the immobilization procedure, in other words is the spectrum obtained free from interfering background signals?

3) Sensitivity: are there enough receptors per mm to bind an amount of ligand that is above the detection limit?

4) Is it possible to make a suitable preparation on the MALDI target with matrix and membrane fragments immobilized on the target surface, in such a way that a spectrum is obtained?

5) Does non-specific binding occur, and to what extent?

6) Does the invention lead to the selective detection of a N2 ligand, i.e. are peptides which do not bind to the N2 receptor really not detected after washing?

This example is an application of the invention in the field of combinatorial chemistry for the purpose of drug discovery. 2. Materials and methods

The experiments were carried out on a Bruker BiFlex III MALDI-ToF-MS fitted with an N₂ laser (337 nm, ~3 ns pulses, diameter of the laser beam about 100 μm). The target used was a SCOUT 384 having a gold layer. All measurements were performed with positive ion detection. The mass calibration of the BiFlex III was done with a mixture of Angiotensin I, Angiotensin II, Bombesin, ACTH 18-39 and Somatostatin (all peptides). Using the procedures described, a N2 receptor coating was applied to the sample spots on the MALDI target. Experiments with the β₂ receptor were also carried out in order to study any non-specific interactions of ANP. In addition to ANP, three other peptides were tested, namely Bombesin, ACTH 18-39 and Angiotensin I. These 3 peptides do not bind to the N2 receptor, at any rate not via a specific ligand- receptor interaction. The molecular masses of these components are:

Angiotensin I 1295.7 ANP 1084.4

Bombesin 1618.8 ACTH 18-39 2465.2

With MALDI all give an [M+H]⁺ ion with an m/z which is 1 higher than the stated molecular masses.

The following solutions were then pipetted onto the sample spots (both for V2 and β₂):

1) blank solvent (100 mM TRIS / 10 mM MgCl₂ in 0.2% (w/v) BSA solution)

2) ANP (2 μl, 1 μmol 1, total 2 pmol absolute)

In order to test the selectivity, a mixture of ANP, Bombesin, Angiotensin I and ACTH 18-39 was applied to sample spots with immobilized N2 receptor.

All the experiments were performed in triplicate, i.e. 3 sample spots per experiment.

The target was then rinsed with a 10 mM TRIS/1 mM MgCl₂ pH 7.5 solution in order to remove the components not binding to the receptor. In theory, only receptor-bound ANP was now present on the sample spots where the N2 receptor was immobilized.

After evaporation of the solvent under nitrogen, the sample spots were prepared for MALDI measurements by application of 1 μl of a DHB solution (2,5-dihydroxy- benzoic acid 20 mg/ml, the solvent was 0.1% trifluoroacetic acid in water/acetonitrile (2/1 v/v)). The sample spots were dried in the ambient air without heating. It was also tested whether the measurements could also be performed without using the DHB matrix.

3. Results and discussion

Figures 5, 6 and 7 show the mass spectra obtained for:

1) N2 receptor with ANP, Angiotensin I, Bombesin and ACTH 18-39

2) β₂ receptor with ANP (top)

3) N2 receptor with ANP (bottom)

The spectra were obtained under identical conditions (output of the laser, number of summed spectra).

The results show clearly that:

1) In the case of a mixture of a ligand (ANP) and non-ligands, only ANP is detected.

2) The receptor-ligand interaction is broken by MALDI. It is not known whether this is already a consequence of the addition of DHB (plus TFA and acetonitrile) or is induced by the laser pulse. This is in any case not relevant for the principle of operation and the applications of the invention.

3) Good spectra are obtained with the standard DHB sample preparation. The observed signal from ANP is identical to that from a standard. The results of the measurements without DHB indicate that the use of matrix is essential.

4) The background is clear, the signals originating from the membrane fragments and the immobilization are only observed at a low m z.

5) In addition to specific binding, non-specific binding also occurs (ANP signal in Figure 6). It is in fact the case that the intensity of ANP relative to the background signals is considerably lower for the β₂ receptor than for immobilized N2 receptor. Sample spots with N2 receptor gave an ANP signal, irrespective of the position of the laser pulse. For the β₂ receptor, however, it is necessary to look for 'sweet spots' in order to detect ANP.

The conclusion is that the invention works in accordance with the theory and that it can be used for the stated applications and possibly other applications based on the principle of receptor affinity. The successful application is only dependent on an optimal washing procedure to remove compounds which bind non-specifically from the sample spot, while the compounds which bind specifically are not removed. In addition to receptor affinity, the invention is also applicable for any other interaction between a compound and the immobilized membrane (blockers of transport channels in the membrane, binding to membrane-bound antigens, etc.).

References

1 Krone, Nelson, Dogruel, Williams, Granzow, Anal. Biochem. 1 Jan 1997, 244(1), 124-132.

2. Nelson, Jarvik, Taillon en Tubbs, Analytical Chemistry, 15 Jul 1999, 71(14), 2858-2865.

3. Dalmasso, ACCIS-Newslet. Oct 1999 (595), 8-10.

Claims

1. Method for reversibly linking a specific binding substance, such as a receptor, to a sensor surface, which receptor is present in cell membrane fragments, characterized in that the receptor is joined to a coupling substance which can enter into a specific and reversible bond with an antibody, and the antibody is joined to the sensor surface.

2. Method according to Claim 1, in which the coupling substance is an antigen and the antibody is an antibody of the antigen.

3. Method according to Claim 1 or 2, in which the coupling substance is biotin and the antibody is antibiotin.

4. Method according to any one of the preceding claims, in which the antibody is joined to the sensor surface by means of a covalent bond.

5. Method according to any one of the preceding claims, in which the receptor is joined to the coupling substance by means of lectin bound to the coupling substance.

6. Method according to any one of the preceding claims, in which at least 10⁹, in particular at least 10 , receptors are linked per mm of sensor surface.

7. Method according to any one of the preceding claims, in which the sensor surface is activated by plasma etching, followed by reaction with a bifunctional crosslinker, before the antibody is joined to the sensor surface.

8. Method according to any one of the preceding claims, in which, prior to the linking of the receptor and sensor surface, a used sensor so linked is regenerated by incubation or injection with a regeneration liquid.

9. Sensor which is reversibly coated with a natural receptor, obtainable by the method according to any one of the preceding claims.

10. Method for determining specific binding between a natural receptor and a ligand, characterized in that the sensor coated with the receptor according to Claim 9 is brought into contact with the ligand and the interaction between the receptor and the ligand is measured.

11. Method for identifying a ligand which has a specific bond with a natural receptor, characterized in that the sensor coated with the receptor according to Claim 9 is brought into contact with the ligand and the ligand, which may or may not be still in contact with the sensor, is identified.

12. Method for isolating, from a mixture, a ligand which has a specific bond with a natural receptor, characterized in that the sensor coated with the receptor according to Claim 9 is brought into contact with the ligand in the mixture, the sensor is separated from the mixture, and the ligand is separated from the receptor.

13. Method according to Claim 10 or 11 , in which the interaction is measured and the identification is performed, respectively, by means of Mach-Zehnder interferometry or another optical detection method based on evanescent field measurements.

14. Method according to Claim 10 or 11, in which the interaction is measured and the identification is performed, respectively, by means of mass spectrometry.

15. Method according to Claim 14, in which Matrix- Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry is used.