WO2001006236A1

WO2001006236A1 - High throughput analysis of molecular interaction using surface plasmon resonance

Info

Publication number: WO2001006236A1
Application number: PCT/EP2000/006710
Authority: WO
Inventors: David Bancroft; Stephan Schmidt
Original assignee: Gpc Biotech Ag
Priority date: 1999-07-15
Filing date: 2000-07-13
Publication date: 2001-01-25
Also published as: DE19933212A1; AU6563800A

Abstract

Apparatus and method for the detection of molecular interactions between a plurality of n different molecular species Si(a) (1 ≤ a ≤ n) and a further plurality of m molecular species Sg(b) (1 ≤ b ≤ m), wherein the Si(a) are immobilized at a phase boundary, and wherein the Sg(b) are comprised in a non-solid phase, in which they are free to move, further comprising: a) a plurality of n + x measuring points Mi(c, a) (1 ≤ c ≤ n+x), x comprising the plurality of molecular species Ki(x) (0 ≤ x ≤ n) of defined properties, serving as control and/or calibration, located at the phase boundary, onto which measuring points the Si(a) have been immobilized in such a manner that to each measuring point molecules of substantially one molecular species Si(a) are immobilized, and that preferably this set of immobilized molecular species are different at the largest part of the measuring points, and b) a detection system, which is capable of measuring a physical property of a phase boundary at a point Mi(c, a) of the phase boundary, which physical property changes with the advent of an interaction between a molecular species Si(a) and/or Ki(x) immobilized at Mi(c, a) and at least one other molecular species Sg(b), for each measuring point Mi(c, a) independently.

Description

HIGH THROUGHPUT ANALYSIS OF MOLECULAR INTERACTION USING SURFACE PLASMON RESONANCE

This invention describes a method and apparatus that allows the analysis of a large number of immobilized molecules for interaction with a number of different molecules by means of surface plasmon resonance (SPR). (Nelson RW, Krone JR. Advances in surface plasmon resonance biomolecular interaction analysis mass spectrometry (BIA/ S). J ol Recognit. 1999 Mar-Apr;12(2):77-93., Nice EC, Catimel B. Instrumental biosensors: new perspectives for the analysis of biomolecular interactions. Bioessays. 1999 Apr;21(4):339-52., Maimqvist M. BIACORE: an affinity biosensor system for characterization of biomolecular interactions. Biochem Soc Trans. 1999 Feb;27(2):335-40.)

The screening of large numbers of molecules (e.g. molecules like proteins, peptides, nucleic acids, oiigosaccharides, phages, small synthetic molecules, antibodies or enzymes) with other molecules by SPR is currently limited to the processing of approximately 200 samples/day. This is due to the sophisticated mechanism of solvent delivery of conventional SPR systems, together with a fixed excitation and detection system designed for 'real time' measurement of interacting molecules. The throughput of these conventional SPR devices is sufficient for in depth secondary or tertiary investigations of a small number of known sets of interacting molecules where for example, kinetic and stoichiometric information of the interaction are to be determined.

SPR is a highly sensitive technique that in principle would be highly suitable for use in primary screening of interactions. That is, the initial identification of those molecules that interact with one or more given molecules. The art would greatly benefit from an invention that was able to screen hundreds or thousands of molecules for potential interaction in a short period of time. One field of application being the high-throughput screening (HTS) of libraries of small molecule compounds for interaction/binding to potential drug targets - where the libraries can number several 100's of 1000's of compounds in size. Another application is genome-scale identification and/or investigation of protein-protein, protein-peptide and peptide-peptide interactions. Yet another application is the investigation of differential protein expression directly from tissue samples where typically 1000's or 10' s of 1000's of different proteins are expressed per tissue. However, application of conventional SPR approaches are unable to match the throughout requirements for such investigations. In this invention we disclose methods whereby it is possible to screen for molecular interactions with a much higher throughput using a SPR-detection device. Furthermore, we disclose apparatus suitable for high throughput screening of molecular interactions.

The apparatus disclosed by this invention comprises:

1. A plurality of molecules of interest deposited as distinct spots in an ordered fashion directly on a pre-activated SPR-chip (or SPR-surface). Said plurality may be between 1 and 10,000 distinct spots per cm², where methods to create such densities are well known in the art. The distance between the immobilized spots may be optimized in order to minimize or prevent signal-interference between the spotted molecules. It is advantageous to designate several spots on the chip as 'controls', where said controls spots may comprise a background, positive or negative control molecules (Figure 1).

2. A fluidic system designed such that a multitude of molecules of interest are exposed to the same fluid. In one embodiment of the invention, a single fluidic channel is used to expose in parallel a fluid to all sample molecules positioned on the chip. In another embodiment of the invention, particular sets of molecules are exposed to one or more different fluids controlled through multiple fluidic channels, which may be co-controlled by valves linking different fluidic channels together. In yet another embodiment of the invention, said fluidic system is separated in time and space from the SPR detection device.

3. The novel SPR chip is read by a modified SPR detection device, whereby the SPR signal (angle, wavelength or refractive index) is separately measured for each distinct molecule spot. This can be enabled for example, by systematically moving the chip using a precision X-Y positional-controller such that each distinct molecule spot is sequentially positioned under the detection point. Said precision X-Y positional-controllers are well known in the art for example, of microscopy and ALDI mass-spectrometry. In another embodiment of the invention, the SPR signal is rapidly detected for each distinct spot by for example, utilizing a laser-scanning system such as that found in laser-scanning fluorescent microscopes (Figure 2). 4. In each of these embodiments of the invention, the collection of data comprising the measured SPR signal for each distinct spot on the chip is stored in a computer readable memory to allow for subsequent data analysis. It is preferable that said collection of data is stored, displayed and analyzed as a digital image. Within said digital image, the relative position of a distinct spot from the chip is represented by the position of a given pixel in the image and the magnitude of SPR signal measured for that spot represented by the value of said given pixel.

One method disclosed by this invention for the high throughput identification of interacting molecules comprises the steps:

1. Optional calibration of a SPR chip by using a SPR-detection device such as described above to read the whole chip before applying the molecules in order to create a "blank" data set. This step may be necessary to occasionally calibrate the system or quality control the SPR-chips before deposition of distinct molecule spots.

2. After deposition of the molecules as distinct spots on the SPR-chip, data comprising the measured SPR signal for each distinct spot on the chip is collected and stored as previously described to create a reference set of SPR signals for each molecule prior to any potential interaction with other molecules. 3. The arrayed chip is then flushed and incubated in a batch mode with the solution containing the potentially interacting molecule(s) and washed to remove unspecific bound material from the chip surface.

4. After washing, data comprising the measured SPR signal for each distinct spot on the chip is collected using the SPR detection device and stored as previously described to create an experimental data set. Said experimental data set records changes in the SPR-signal, which is related to the size and amount of interacting molecule bound to the first immobilized molecule.

5. Optionally, those steps of incubating, washing and measuring can be repeated several times in order to create a complex sandwich on interacting molecules.

6. It may be advantageous to conduct the binding, incubation and/or washing steps separately from the SPR-detection device by removing the SPR-chip from the SPR-detection device and conducting the binding, incubation and/or washing steps in a separate apparatus and then replacing the SPR-chip into the SPR-detection device to measure the SPR-signal. In this way, a larger number of chips and hence samples may be processed for a given SPR- detection device. The absolute change in SPR-signal measured for a given spot may change simply because it has been removed and replaced into the SPR-detection device, or has been measured using two different SPR- detection devices. Therefore, a normalized SPR-signal and hence data set may be calculated using data for appropriate internal control spots on the chip as described above.

7. Changes in the SPR-signal for each individual spot are detected by comparison of the experimental or normalized data set collected after each additional incubation with the reference data set. A significant change in SPR signal for a given spot identifies that the molecule deposited at that position has interacted with the potentially interacting molecule.

8. Following this efficient primary screen, those molecules showing significant change in SPR-signal may be further investigated for their interaction with the potentially interacting molecule using for example, conventional SPR methods, Mass-Spectrometry or other analytical methods. Another method disclosed by the invention is the investigation of differential protein expression directly from tissue samples comprises the steps:

1. Providing a SPR chip comprising a multitude of antibodies that bind proteins of interest. To assist in the subsequent interpretation of data, it is advantageous to have control regions and/or control antibodies also present on the chip. Optionally, the antibody chip is calibrated as described above.

2. Incubating said antibody SPR-chip with a crude protein extract made from the first tissue sample so as proteins in the sample bind to antibodies on the array. Non-specific binding is minimized by subsequently flushing the chip with a washing fluid. Optionally, these steps may be conducted separately from the SPR-detection device.

3. The SPR signal for each antibody spot of the chip is then measured and recorded as described above. Optionally, the raw experiential data may be normalized by consideration of the SPR signals derived from the control regions of the chip. The SPR signal detected for a given antibody spot is a measure of the amount of antibody and protein from the first sample tissue bound to the antibody at that spot position.

4. After recording SPR data for each antibody of the array following binding with proteins of the first tissue sample, the bound proteins are removed from the chip by an appropriate stripping and regeneration procedure. Optionally these steps may be conducted separately from the SPR-detection device. Alternatively, an essentially identical or calibrated second SPR chip is used to replace the first.

5. A crude protein extraction of a second tissue sample is then flushed and incubated such that said second set of proteins bind to the antibodies on the SPR chip. The chip may be washed to remove non-specific binding. These steps may optionally be conducted separately from the SPR-detection device. The new SPR signal is collected for each spot on the chip to record the amount of protein from the second tissue sample bound to the antibodies to give a second experimental or normalized data set as described above. 6. Optionally, the SPR chip can be regenerated or the chip replaced once more and protein samples from further tissue samples analyzed for protein expression as described above.

7. Changes in the SPR-signal for each individual antibody spot are detected by comparison of the experimental or normalized data set between two or more tissue samples. A significant change in SPR-signal between two tissue samples for a given antibody spot identifies that a protein able to bind to said individual antibody spot was expressed at different levels in said two tissue samples. By investigation of potential changes in SPR signal or multiple antibody spots, the protein expression pattern for a given tissue can be determined.

Example:

(All buffers, solutions or procedures without explicit reference can be found in standard textbooks, for example Coico R., Current Protocols of Immunology, John Wiley and Sons, 1999 or Sambrook J., Fritsch E.F., Maniatis T., Molecular Cloning: A Laboratory Manual, CSHL-Press, 1989.)

A) Preparation of a molecule array Arrays of a plurality of molecular species are prepared either by covalent attachment according to available functional groups, e.g. -SH, -NH₂, -COOH, -CHO, or by fixing the respective molecule by a specifically affinity support (e.g. biotinylated sample on streptavidin matrix, or antibodies on a protein G support) which is well know in the art. It is advantageous to create a uniform affinity support, which allows a simple one step affinity based fixation of molecules which all belong to the same class of molecule (e.g. all are different antibodies) onto that surface, and ideally allow directed and uniform spatial orientation. Procedures to generate an affinity support are identical to the methods to generate covalent attachments via functional groups. The transfer of a multitude of different molecules to discrete regions on the support is achieved by using the fluidic system of a Biacore 2000® of Biacore AB, Uppsala Sweden, according to standard procedures. Other methods of creating discrete regions on a SPR support is achieved by use of a custom SPR chip such as described in WO99/60382, where each tooth of a comb is used to bind to a discrete molecular species used in a discrete chamber.

Alternatively, the transfer of multiple molecular species to discrete regions on the support are well known in the are, for example by spotting devices, (Lueking A, Horn M, Eickhoff H, Bussow K, Lehrach H, Walter G. Protein microarrays for gene expression and antibody screening. Anal Biochem 1999 May 15;270(1):103-11) or nanoliter dispensing pipettors. A standard chip of Biacore with the dimensions of 6 mm x 6 mm loaded with a robotic spotter as described above allows the immobilization of more than 200 different samples on said chip. Other procedures how to generate arrays of chemical and biological molecules are well known in the art.

B) Repeated external incubation and internal measurement In order to demonstrate the feasibility of external treatment of the chip we removed and reinserted the chip from a SPR-device (Biacore 2000® of Biacore AB, Uppsala Sweden,) and compared the SPR-signal for a given spot obtained before and after reinsertion. Surprisingly we found that the SPR-signal for a given spot recorded shortly after completion of chip docking, was similar to the SPR-signal recorded for the same spot before removal and reinsertion (Figure 3). This observation lead to the conclusion that it is possible to incubate the whole chip containing an immobilized plurality of molecular species externally with another plurality of molecules, potentially interacting with the first group of molecules, which bind to their corresponding immobilized partners without using a sophisticated flow system as in the case of the Biacore device. The incubations with interacting molecules are performed either simultaneously with a mixture of molecules in a bathing or spraying device or sequentially one after another with a pumping system (Figure 4). Likewise we observed that washing and regeneration steps may be conducted separately from the SPR-detection device, and allow the collection of reliable and/or comparable SPR- signals after reinsertion and measurement using the SPR-detection device. C) High Throughput-binding assays We used this surprising element to demonstrate the utilization of the invention for high throughput binding assays as follows:

A chip is prepared according to example A and incubated with a plurality of molecular species externally. The external incubation with a plurality of molecular species simultaneously results in a dramatic speed increase of the whole process, which is a substantial advantage compared to measurements in flow systems. Working with the number of 200 individual samples as in the example A the speed increase is in the range of 50 fold compared to a 4 position chip. The speed of the whole process is only defined by the speed of the measuring within the apparatus, since a multitude of chips can be handled in parallel to generate the matrix of measuring points with immobilized molecular species as well as the incubation with a multitude of samples that are incubated with the prepared chips. Therefore an even greater increase of throughput is achieved.

Such a technology is for example useful for the readout of an antibody array incubated with a complex mixture of different molecules, such as creating a specific interaction map which is characteristic for the analyzed sample. Standardized arrays can be produced and may be reused for a number of screens

Further analysis of spots with interacting molecules attached to it can be performed with for example, mass-spectrometric devices, which either use the unmodified chip with immobilized molecules or the chip following selective stripping of the array.

Claims

Claims:

1. Apparatus for the detection of molecular interactions between a plurality of n different molecular species Sj(a) (1 < a < n) and a further plurality of m molecular species S_g(b) (1 < b < m), wherein the Sj(a) are immobilized at a phase boundary, and wherein the S_g(b) are comprised in a non-solid phase, in which they are free to move, further comprising a. a plurality of n + x measuring points Mi(c,a) (1 < c < n+x), x comprising the plurality of molecular species Kι(x) (0 < x < n) of defined properties, serving as control and/or calibration, located at the phase boundary, onto which measuring points the Sj(a) have been immobilized in such a manner that to each measuring point molecules of substantially one molecular species Si(a) are immobilized, and that preferably this set of immobilized molecular species are different at the largest part of the measuring points, and b. a detection system, which is capable of measuring a physical property of a phase boundary at a point Mj(c,a) of the phase boundary, which physical property changes with the advent of an interaction between a molecular species Si(a) and/or Kj(x) immobilized at Mj(c,a) and at least one other molecular species S_g(b), for each measuring point Mι(c,a) independently.

2. Apparatus according to claim 1 wherein it is possible to let occur the interaction between the molecular species S,(a) and/or Ki(x) immobilized at the phase boundary at Mj(c,a) and at least one further molecular species S_g(b) independently from the detection system and the detection with regard to space and time.

3. Apparatus according to claim 1 and 2, wherein the phase forming the phase boundary which does not comprise the S_g(b) is a solid phase.

4. Apparatus according to claims 1 to 3 wherein said detection system comprises a light source and a light detector.

5. Apparatus according to any one of claims 1 to 4 wherein said physical property is the SPR signal (angle, wavelength, refractive index) at the optical surface formed by the phase boundary.

6. Apparatus according to any one of claims 1 to 5 wherein the plurality of n molecular species Sj(a) immobilized at the phase boundary exceeds 4 different Si(a), preferably exceeds 10 different Sj(a), more preferably exceeds 50 different Sj(a), most preferably exceeds 100 different Sj(a).

7. Phase boundary preferably for use in an apparatus according to any of claims 1 to 6 where the plurality of n molecular species Sj(a) immobilized at the phase boundary exceeds 4 different Sι(a), preferably exceeds 10 different Sj(a), more preferably exceeds 50 different Sj(a), most preferably exceeds 100 different

8. Apparatus to allow interaction between the molecular species Sj(a) and/or Ki(x) immobilized at the phase boundary at Mi(c,a) and at least one further molecular species S_g(b) comprised in a non-solid phase, in which said further molecular species S_g(b) are free to move.

9. Apparatus according to claim 8, which allows the washing and/or regeneration of the phase boundaries.

10. Apparatus according to claims 8 or 9 wherein interaction, washing and/or regeneration occurs by using fluidic processes comprising pumping, spraying and/or bathing.

11. Apparatus according to any one of claims 1 to 10 wherein at least one SPR- signal measured from said molecular species Kj(x) (0 < x < n) of defined properties are used to normalize data collected from the measurement steps.

12. Apparatus according to any one of claims 1 to 11 wherein the physical property measured or changes with the advent of an interaction between a molecular species Si(a) or Kι(x) immobilized at Mi(c,a) and at least one other molecular species S_g(b), for each measuring point Mj(c,a) are displayed as image.

13. Apparatus according to any one of claims 1 to 12 wherein said Si(a) and S_g(b) and Ki(x) belong to one or more of the molecular classes of proteins, peptides, nucleic acids, carbohydrates, lipids, small organic and inorganic synthetical chemical compounds, natural product extracts, whole cells of bacteria, fungi or viruses.

4. Method for the detection of molecular interactions between a plurality of n different molecular species S,(a) (1 < a < n) and a further plurality of m molecular species S_g(b) (1 < b < m), wherein the Sι(a) are immobilized at a phase boundary, and wherein the S_g(b) are comprised in a non-solid phase, in which they are free to move, further comprising c. immobilizing a plurality of n + x measuring points Mi(c.a) (1 < c ≤ n+x), x comprising the plurality of molecular species Kj(x) (0 < x < n) of defined properties, serving as control and/or calibration, located at the phase boundary, onto which measuring points the Si(a) have been immobilized in such a manner that to each measuring point molecules of substantially one molecular species Sj(a) are immobilized, and that preferably this set of immobilized molecular species are different at the largest part of the measuring points, and d. measuring a physical property of a phase boundary at a point Mj(c,a) of the phase boundary, which physical property changes with the advent of an interaction between a molecular species Sj(a) and/or Ki(x) immobilized at Mj(c,a) and at least one other molecular species S_g(b), for each measuring point Mj(c,a) independently, and e. letting occur the interaction between the molecular species Sι(a) and/or Ki(x) immobilized at the phase boundary at Mi(c,a) and at least one further molecular species allowing the interaction between Sj(a) and S_g(b), and f. measuring a physical property of a phase boundary at a point Mj(c,a) of the phase boundary, which physical property changes with the advent of an interaction between a molecular species Si(a) and/or Ki(x) immobilized at Mj(c,a) and at least one other molecular species S_g(b), for each measuring point Mj(c,a) independently, and g. detecting a molecular interaction between a molecular species Sj(a) and/or Ki(x) immobilized at Mj(c,a) and at least one other molecular species S_g(b), for each measuring point Mi(c,a) by comparing the values of said physical property of a phase boundary at a point Mι(c,a) of step d and f, at which a change in the measured value of said physical property between step d and f indicates a molecular interaction.

15. Method according to claim 14 wherein the interaction between the molecular species Sι(a) and/or Ki(x) immobilized at the phase boundary at Mi(c,a) and at least one further molecular species S_g(b) occurs independently from the detection system and the detection with regard to space and time

16. Method according to claim 14 or 15 where the values or change of said physical property collected from a plurality of measuring points Mj(c,a) in steps d, f and/or g are displayed as an image.

17. Method according to any one of claims 14 to 16, wherein the phase forming the phase boundary which does not comprise the S_g(b) is a solid phase.

18. Method according to any one of claims 14 to 17 wherein said measurement of the physical property of a phase boundary in step d and/or f uses a light source and a light detector.

19. Method according to any one of claims 14 to 18 wherein said physical property is the SPR signal (angle, wavelength, refractive index) at the optical surface formed by the phase boundary.

20. Method according to any one of claims 14 to 19 wherein the plurality of n molecular species Sι(a) immobilized at the phase boundary exceeds 4 different Si(a), preferably exceeds 10 different Sj(a), more preferably exceeds 50 different Sj(a), most preferably exceeds 100 different Sj(a).

21. Method according to any one of claims 14 to 20 wherein the phase boundary is washed and /or regenerated independently from the detection system with regard to space and time.

22. Method according to any one of claims 14 to 21 wherein said interaction, washing and/or regeneration occurs using fluidic systems comprising pumping, spraying and bathing.

23. Method according to any one of claims 14 to 22 wherein said Sj(a) and S_g(b) and Ki(x) belong to one or more of the molecular classes of proteins, peptides, nucleic acids, carbohydrates, lipids, small organic and inorganic synthetical chemical compounds, natural product extracts, whole cells of bacteria, fungi or viruses.

24. Method according to any one of claims 14 to 23 wherein at least one SPR-signal measured from said molecular species Kj(x) (0 < x < n) of defined properties are used to normalize data collected from the measurement steps.

25. Use of an apparatus according to any one of claims 1 to 13 to identify interacting molecular species.

26. Use of a method according to any one of claims 14 to 24 to identify interacting molecular species.

27. Use of an apparatus according to any one of claims 1 to 13 and/or a method according to any one of claims 14 to 24 to identify interacting molecular species wherein said species belong to one or more of the molecular classes of proteins, peptides, nucleic acids, carbohydrates, lipids, small organic and inorganic synthetical chemical compounds, natural product extracts, whole cells of bacteria, fungi or viruses.

28. Method for the preparation of a formulation, which is characterized by containing at least one step comprising h. the use of an apparatus according to any one of claims 1 to 13, a method according to any one of claims 14 to 24 or the use of apparatus or method according to any one of claims 25 to 27 to detect an interaction, which is relevant for a particularly favorable or unfavorable physical condition, and i. the identification of an agonist, inhibitor or antagonist by the interaction which is described in step h.

29. Method for the identification of an antagonist of an interaction identified according to any one of claims 1 to 27 or a method comprising j. the use of an apparatus according to any one of claims 1 to 13, a method according to any one of claims 14 to 24 or the use of apparatus or method according to any one of claims 25 to 27 to detect an interaction, which is relevant for a particularly favorable or unfavorable physical condition, and the identification of an agonist, inhibitor or antagonist by the interaction which is described in step j.