EP1514135A1

EP1514135A1 - Differential indication of labeled molecules

Info

Publication number: EP1514135A1
Application number: EP03735530A
Authority: EP
Inventors: Wojciech Wozny; Michael A. Cahill
Original assignee: ProteoSys AG
Current assignee: ProteoSys AG
Priority date: 2002-06-03
Filing date: 2003-06-03
Publication date: 2005-03-16
Also published as: AU2003238205A1; US20050176012A1; WO2003102627A1; DE10225841A1; JP2005528623A

Abstract

The invention relates to a method for determining the local distribution of at least two sets of point radiation objects of a quantity-specific radiation type on a common support. According to a preferred embodiment, a phosphorimager IP is subjected to the radiation of the planar support during 24 hours. The phosphorimager IP integrates the radiation intensities of both radiation sources of the samples. The local distribution of the stored radiation energy of the phosphorimager IP is read with a reader and represented as a pixel matrix or an image. The local distribution determined as a pixel matrix has portions that are derived from I<125> and I<131>. Once this first local distribution is determined, a second local distribution is determined, whereby an absorber is disposed between the planar support and the phosphorimager IP. The absorber consists of a 900 pm thick plastic layer. The radiation intensities of the samples disposed on the planar support are integrated or added up for both local distributions determined using the same ranges. Starting from these integrated radiation sources, the local distributions already determined are used to separately calculate the local distributions of the individual isotopes. The inventive method is especially used for analyzing protein gels, in nucleic acid arrays, protein arrays, ELISA analysis etc. in proteomics.

Description

Differential display of labeled molecules

Field of application and state of the art

The invention relates to a method for determining the spatial distribution of at least two sets of point radiation objects, each with a set-specific radiation type, on a common carrier surface.

An important application of proteomic studies is to determine the difference in the frequencies of certain proteins or protein isoforms between two or more experimentally interesting regimes, for example the frequency of proteins from control cells, tissues or organisms, in comparison to the corresponding frequency from pharmacologically treated cells, tissues or organisms.

The protein intensity for protein spots from different gels from different experimental regimes in the study are typically compared to determine whether the amount of protein in these protein spots differs between the experimental regimes. This is a principle of differential representation, which is also referred to as multicolour analysis. The assignment of protein spot coordinates and the identification of corresponding protein spots on different 2D gels is called the matching procedure and is potentially problematic because of the non-reproducible behavior of different gels that are generated by 2D PAGE. Therefore, the matching process is a time-consuming step and can currently only be automated to a limited extent.

For example, a rough multicolor analysis can be performed by measuring the different samples in different experimental systems be performed. For example, proteins from different samples can be treated in separate 2-D-PAGE experiments, followed by detection of the spots that correspond to proteins in the different 2D-PAGE gels, and comparison of those for each sample of each gel or each Group of replicated gels obtained spot patterns. The spot patterns are typically stored in graphic gel image files, such as e.g. B. TIFF files. Conventional software program algorithms align these gel image files with each other so that pixels in all images can be compared with pixels from other images. Typically, a "composite gel image" is made from replicated 2D PAGE gel images for a sample that contains averages of the spot intensities of all replicate gels for a particular sample. The composite gels from different samples are typically combined with each other. The two main limitations of this approach are spot matching, which enables the correct allocation of the corresponding spots and the quantitative evaluation of the spots, which enables a comparison of the protein amounts in spots from different 2D gels ,

In order to produce a finer differential representation, analyte molecules from different samples are typically marked or modified with reagents so that the samples can be mixed and analyzed together. In this way, the complex and error-prone matching process can be avoided. The proteins of each sample can then be detected based on their labeling regardless of the presence of the proteins from the other sample (s).

One example is the DIGE fluorescent reagents based on Cye dyes, which are offered by Amersham Biosciences (Freiburg, Germany). Proteins from both samples are - -

fluorescence reagents are labeled separately, then mixed and subjected to the electrophoresis in 2D-PAGE. The different fluorescent groups in each sample are excited by different wavelengths and emit light at different wavelengths. By using suitable light filters, it is possible to obtain images of proteins from each sample, regardless of the wedging of the proteins from the other sample. These images can then be analyzed using appropriate software packages.

Another approach for a finer differential representation is the labeling of analyte molecules from different samples with radioactive isotopes. The radioactive labeling of analyte molecules and their detection by suitable detectors is several orders of magnitude more sensitive than other methods, for example the method mentioned above using Cye dyes.

An analysis method in which analyte molecules are labeled with radioactive isotopes is described in:

Monribot-Espagne C, Boucherie H, Differential gel exposure, a new methodology for the two-dimensional comparison of protein samples. Proteomics, 2002, 2: 229-240.

When labeling analyte molecules with radioactive isotopes, it is necessary to determine the radioactive radiation or the activity of the isotopes. The nuclei of radioactive isotopes are unstable. They tend to disintegrate spontaneously with the release of particles or photons. Radioactive substances release several different types of particles. The electron, the positron, the alpha particle and the neutron are of general importance. The emission of these particles is often, but not always, accompanied by the emission of gamma rays. Another type of radioactive decay is the spontaneous capture of an electron by the K shell the core of what is known as K electron capture. Radiation detectors are devices that have been developed to detect these different types of radiation.

The following spatially resolving detectors are used for the quantitative analysis of radioactive molecules present in an array or on a surface such as a DNA array or a 2D PAGE protein gel and replace a photographic film for this purpose. Of the four examples cited, detector types 2, 3 and 4 are based on the above principles.

1. Autoradiography with phosphor imagers that operate according to the principles of devices that use an image plate, such as those currently available from Fuji Photo Film Co. Ltd (Tokyo), Packard Bioscience GmbH (Dreieich Germany), AGFA-Gevaert N.V. (Mortsel, Belgium) or Amersham Biosciences (Freiburg, Germany). Amemiya Y, Miyahara J. Imaging plate illuminates many fields. Nature, 1988, 336: 89-90.

Miyahara, J. The imaging plate: a new radiation image sensor. Chemistry Today, 1989, 223: 29-36.

2.PPAC (parallel plate avalanche chamber) ionization gas chambers, which are coupled to microchannel plate analyzers for the detection of beta particles, such as the beta imager by BioSpace Measures (Paris) and a similar device by Packard Bioscience ( Dreieich, Germany).

In these imagers, light from the PPAC is focused through a lens into a multi-fiber image intensifier tube, from which the signal is captured by a CCD camera. Laniece P, Charon Y, Dumas S, Mastrippolito R, Pinot, L, Tricoire H, Valentin L. HRRI: a high resolution radioimager for fast, direct quantification in in situ hybridization experiments. Biotechniques, 1994, 17: 338-345.

Crumeyrolle-Arias M, Latouche J, Laniece P, Charon Y, Tricoire H, Valerien L, Roux P, Mirambeau G, Leblanc P, Fillion G, et al. "In situ" characterization of GnRH receptors: use of two radioimagers and comparisons with quantitative autoradiography. J. Recept Res, 1994, 14: 251-265.

Jeavons, AG. Method and apparatus for quantitative autoradiography analysis. 1 August 1, 1992. U.S. Patent 5138168. Owner: Oxford Position Systems Limited.

Decristoforo C, Zaknun J, Kohler B, Oberladstaetter M, Riccabona G. The use of electronic autoradiography in radiopharmacy. Nucl. Med. Biol, 1997, 24: 361-365.

3. Devices such as the Micro Imager (BioSpace Measures, Paris), in which a scintillation surface for the detection of beta-gamma and alpha particles is coupled with micro-channel plate analyzers. In these imagers, light from the scintillation surface enters a multi-fiber image intensifier tube, from which the signal is captured by a CCD camera.

Laniece P, Charon Y, Dumas S, Mastrippolito R, Pinot, L, Tricoire H, Valentin L. HRRI: a high resolution radioimager for fast, direct quantification in in situ hybridization experiments. Biotechniques, 1994, 17: 338-345.

Crumeyrolle-Arias M, Latouche J, Laniece P, Charon Y, Tricoire H, Valentin L, Roux P, Mirambeau G, Leblanc P, Fillion G, et al. "In situ" char- acterization of GnRH receptors: use of two radioimagers and comparisons with quantitative autoradiography. J. Recept Res, 1994, 14: 251-265.

Decristoforo C, Zaknun J, Kohler B, Oberladstaetter M, Riccabona G. The use of electronic autoradiography in radiopharmacy. Nucl. Med. Biol. 1997, 24: 361-365.

4. Scintillation crystal array imagers that use a spatially resolving photomultiplier, such as the MPD imagers (Multi Photon Detection), which are sold by BioTraces Inc. (Herndon VA, USA) and ProteoSys AG (Mainz, Germany).

Drukier AJ, Sagdejev IR, Ultralow background multiple photon detector. February 2, 1999. U.S. Patent 5,866,907. Owner: BioTraces, Inc. (Herndon, VA).

In contrast to the above detector types 2, 3 and 4, a phosphor imager plate (phosphor imager plate) is a film-like radiation image sensor that contains specially designed phosphor that absorbs and stores the radiation energy so that it can be released again at a later time and can be measured in a suitable phosphor imager reader. A phosphor imager is a combined system consisting of a phosphor imager and a phosphor imager, hereinafter referred to as phosphor imager. A phosphor is a powder or a crystalline substance that emits light after exposure to photons of certain properties or chemical reactions. Light emission (luminescence) from a phosphor can be instantaneous (fluorescence), delayed (phosphorescence) or photostimulated Lu- minescence (PSL). The phosphor of the phosphor imager IP uses the PSL phenomenon, which is neither fluorescence nor phosphorescence. PSL uses a substance that stores the energy of an original stimulation, for example the energy of a photon that is emitted by a radioactive decaying atom, in the electron orbitals of atoms of the phosphorus. This energy is emitted as light when the phosphor is stimulated by light with a longer wavelength than that of the first stimulation. In this way, the phosphor stores information about the amount of radiation to which it has been exposed and emits this information as light that can be quantitatively analyzed in a suitable device, such as a commercially available phosphor imager.

The irradiation of samples in the phosphor imager is carried out in a similar manner to the exposure of a photo film. The irradiated phosphor imager IP is scanned during transport with a focused laser beam (for example a He-Ne laser). The PSL released when the phosphor crystals are irradiated with the laser light is collected in a photomultiplier tube (PMT, photomultiplier tube) by a light collector and converted into electrical signals.

From the types of radioactive measuring devices as discussed above (phosphor imagers, PPAC ionization gas chamber detectors, scintillation crystal array imagers with a spatially resolving photomultiplier, scintillator area imaged by a CCD camera), scintillation crystal array imagers can use different isotopes using energy and particle type (beta / gamma) by analyzing the detected pulse shape of different emitted particles. PPAC ionization gas chamber detectors (e.g. beta imager) and scintillation areas (e.g. micro imager) imaged by a CCD camera require the detection of the extremely weak beta particles of H-3 in addition to another isotope in order to detect different radioisotopes distinguish as the manufacturer states in his description. These detectors cannot detect proteins labeled with H-3 directly from a 2D PAGE gel because the polyacrylamide matrix absorbs the weak H-3 beta particles. Analyte molecules must be removed from the polyacrylamide gel, for example by blotting on a membrane, so that they can be detected by PPAC ionization gas chamber detectors. This is time-consuming and only works with variable efficiency for proteins with different molecular weights. Analyte molecules labeled with stronger beta-emitting isotopes, such as S-35, can be efficiently visualized through ionization gas chambers while they are still in a polyacrylamide gel matrix and do not need to be blotted. Scintillation crystal array imagers that use a spatially resolving photomultiplier and ionization gas chambers, however, occupy one device for the entire period of the radioactive radiation. Therefore, analyzes, for example in protein or genome studies, which require high throughput are very expensive.

Further conventional differential detection methods are described below, which, however, cannot be used for the differential detection of analyte molecules on a carrier surface.

The quantitative determination of the concentration of two different elements present in a material sample using two measurements with one detector is described in: Sastri et al., Anal. Chem., 1981, 53: 765-770.

With this method, however, only the concentration of the elements in the material sample is calculated, but not their spatial distribution, for example on the sample surface. The method described here is therefore not suitable for the differential analysis of analyte molecules that are labeled with different isotopes. The hyperspectral processing software MultiSpec © from Purdue University (Purdue Research Foundation, West Lafayette, IN) uses spectral measurements of images to extract or identify various features of satellite images. The MultiSpec © concept provides for the spectral occupation of pixels from one image to be broken down into a series of images, all of which are derived from a master image for which the primary data were all recorded simultaneously. In this method, different images derived from this original image are consequently generated by mathematical methods, starting from a master image.

David Landgrebe, Information Extraction Principles and Methods for Multispectral and Hyperspectral Image Data, Chapter 1 of Information Processing for Remote Sensing, ed. by C.H. Chen, published by World Scientific Publishing Co., Ine, 1060 Main Street, River Edge, NJ 07661, USA, 2000.

Because radioactive labeling of analyte molecules is often the means of choice to achieve the analytical performance required in many studies, such as biological studies, and because differential detection of analyte molecules from two or more samples in one analysis is desirable, there is a great deal It is necessary to carry out such a differential detection with radiolabelled analytes or other molecules.

Task and solution

The object of the invention is therefore to provide a method which enables differential analyzes, for example in protein or genome studies, with high analytical performance and high throughput at comparatively low costs and which helps to overcome the disadvantages of the prior art described. This object is achieved by a method having the features of claim 1. Preferred developments of the invention are listed in the dependent claims 2 to 19. The wording of all the claims is hereby incorporated by reference into the content of this description.

In the method according to the invention, the local distribution of at least two sets of point radiation objects, each with a quantity-specific radiation type, on a common carrier surface is determined with the following steps: a) determining a first local distribution of the radiation intensity of the carrier surface, b) changing the radiation intensity with at least one quantity-specific radiation type associated change factor, c) determining at least one second local distribution of the radiation intensity of the carrier surface and d) calculating the local distributions of each of the at least two sets of point radiation objects individually from the first and the at least second determined local distribution.

Local distribution of point radiation objects is understood to mean, for example, the radiation intensity and / or the number of the respective point radiation objects per unit area over the location or over the carrier surface to be examined. The spatial distributions of at least two sets of point radiation objects, each with a set-specific radiation type, are examined. A point radiation object is an object, for example an atom or its isotope and / or a molecule consisting of groups of atoms or isotopes, which emits its specific radiation uniformly or at least idealized uniformly on all sides, ie over the solid angle. The quantity-specific radiation type can be, for example, alpha, beta, gamma and / or x-ray radiation and / or light with a specific wavelength. The at least two sets each consist of point radiation objects with the same, specific radiation type and are common on the support surface. The carrier surface is an essentially flat structure, for example a 2D PAGE gel.

In the first step a) of the method according to the invention, a first spatial distribution of the radiation intensity, i.e. the radiation power prc ^ unit area, the carrier area determined.

In the second step b), the radiation intensity of at least one quantity-specific radiation type with an associated change factor, which is known or can be determined or calculated, is changed. The change can be a reduction or an increase in the intensity of the quantity-specific radiation type. The change factor is given in%, where 0% means a complete suppression of the radiation intensity, 100% a constant radiation intensity and values greater than 100% mean an increase in the radiation intensity.

All intensities of the quantity-specific radiation types, only a part of the intensities of the radiation types or only an intensity of a radiation type can be changed by an associated change factor. The ratio or ratios of the change factors of the quantity-specific radiation types preferably have a value or values not equal to 1, i.e. the change factors of the quantity-specific radiation types advantageously differ from one another.

In a further step c), after the change in the radiation intensities, at least one second spatial distribution of the radiation intensity of the carrier surface is determined. In a fourth step d), the spatial distributions of each of the at least two sets of point radiation objects are calculated individually from the first and the at least second determined spatial distribution.

Based on the determined at least two spatial distributions, the calculation separately determines the spatial distribution of each set of spot radiation objects on the support surface, regardless of the distribution of the other sets of spot radiation objects on the support surface. The separation into the quantity-specific local distributions is not already carried out by the detector, but only by calculation based on the local distributions determined, which represent a superimposition of the detected signals of the individual radiation sources.

In an advantageous development of the invention, the change step b) and step c) for determining the at least second spatial distribution of the radiation intensity of the carrier surface are repeated at least once with a different change factor. This is necessary if more than two sets of point radiation objects are to be analyzed, because in order to determine the individual spatial distribution of a set, at least one local distribution with associated change in the intensity of the set-specific radiation type must be determined.

If only two sets of point radiation objects are analyzed, the accuracy of the method can be improved by such a repetition. This is achieved in particular in that the change factors are realized by different mechanisms, for example with the aid of an absorber and / or subsequently by radioactive decay.

In a further advantageous development of the invention, a determined local distribution of the radiation intensity is determined by an associated one - I j -

Pixel matrix shown, wherein a pixel value of the matrix represents the radiation intensity of an associated location on the support surface.

When determining the spatial distributions of the radiation intensity of the carrier surface, for example with a suitable spatially resolving detector, the spatial distribution of the radiation intensity of the carrier surface is converted into a matrix arrangement of pixel values, whereby the pixel values can be proportional to the radiation intensity determined by the detector. Each pixel is assigned to a defined region of the area.

In a further advantageous development of the invention, the spatial distribution of each of the at least two sets of point radiation objects is determined individually on the basis of the pixel matrices.

In a purely illustrative example, the calculation, when analyzing two sets A and B of point radiation objects and a change factor of 0%, i.e. a complete suppression, for the radiation type of set A and a change factor of 100%, i.e. a steadiness, for the radiation type of the amount B, can be carried out as follows.

By completely suppressing radiation type A in the second determined local distribution, one obtains the superimposed local distribution for radiation type B. By subtracting the second determined local distribution, in which only signal components of the radiation type of quantity B are present, from the first determined local distribution, in which If both signal components are present, the spatial distribution of the radiation type of set A is obtained without signal components of radiation type B.

Using the same or a similar detector, the method mentioned enables a multicolour measurement, ie a separate, overlay-free representation of the local distribution of the individual point radiation sources, enables. Conventional methods require the original single-color images of analyte molecules from each sample for a multicolour display. In the present invention, the single-color images of each isotope are never measured as such, but are calculated from the at least two determined local distributions or images.

In practice, the signal components attributable to the different radiation types are advantageously changed or reduced by a factor of less than 100% or 95%. One possibility for calculating the superposition-free local distribution of the two sets of point radiation objects is explained below.

The following applies to an exemplary pixel value of the pixel matrix of the first determined local distribution: A + B = X (1)

The following applies to the pixel value of the second determined local distribution: MA + NB = Y (2):

In which

A = the signal contribution of radiation type A in the first determined

Local distribution B = the signal contribution of radiation type B in the first determined

Local distribution X = pixel value corresponding to the radiation intensity in the first determined local distribution Y = pixel value corresponding to the radiation intensity in the second determined local distribution

M = change factor for the quantity-specific radiation type

A N = change factor for the quantity-specific radiation type B

A and B are unknown and wanted. X and Y are the measured intensities.

M&N can be determined by calibration or calculation. The ratio of the change factors of two radiation types is defined as the ratio of the change factor of the radiation type with the smaller change factor and that of the radiation type with the larger change factor (M / N with N≥M). The relationships of the change factors for more than two types of radiation are defined accordingly.

The values for A and B can be calculated by solving the system of equations consisting of equations (1) and (2). This calculation can be carried out analogously for all pixels of the pixel matrix.

If the radiation intensity or the signal contribution of a radiation type at a location is known, the number of point radiation objects per unit area at the location can also be calculated, since the radiation intensity and the number of point radiation objects per unit area are proportional to one another.

In a more general case with a number P of sets of point radiation objects, in which it is necessary to determine P location distributions, the problem can be formulated using a matrix representation:

F * X = I

in which: X = a vector with P elements, consisting of the signal contributions of the radiation types of the P sets of point radiation objects in the first determined local distribution

I = a vector with P elements, consisting of the pixel values of the

P determined local distributions

F = a matrix with P * P elements, consisting of the P * P change factors, which were calculated or determined for the P local distributions for the P different amounts of point radiation objects.

The individual signal contributions of the radiation types of the vector X can be calculated by inversion of the matrix F according to the following equation:

X = F ^"1 * I

In a further development of the invention, the spatial distribution of each of the at least two sets of point radiation objects is determined individually on the basis of intensities which result from the summation of pixel values of defined, in particular neighboring, elements of the pixel matrix. Instead of using the algorithm for a pixel-by-pixel analysis as described above, the summed-up pixel intensities of certain areas can also be used, which can be calculated for the first and the second determined spatial distribution. This is particularly recommended in the case of very noisy signals or in those cases in which the spatial distribution of the signals on the detector can be varied for the different sets of spot radiation objects.

Preferably, more measurements of the local distributions are carried out under changed conditions than would be necessary to a minimum. In this case, an optimization process is used to identify the individual To determine the spatial distributions of the point radiation objects. This can improve the accuracy of the method.

In a further development of the invention, a set of point radiation objects consists of at least one radiating, in particular radioactive, type of isotope. For example, 1-125 and / or 1-131 can be used as isotopes.

In a development of the invention, a number of point radiation objects consist of light-emitting, in particular fluorescent, phosphorescent and / or luminescent, substances.

In a development of the invention, at least one calibration point with known radiation is present for at least one set of different point radiation objects at at least one location on the carrier surface. The calibration serves to determine or calculate the respective change factors that are required to calculate the individual local distributions sought.

A calibration point can be a single signal source, for example a single radioactive isotope. The purpose of calibration here is to calculate the absolute or relative percentage of the change or reduction in signal intensities that result from the radiation sources or isotopes examined.

The calibration can be carried out by introducing a radioactive starting material for one or more of the isotopes examined in the measurement. The signal measured by this calibration substance should preferably contain no or negligible proportions from all other radioactive sources outside the calibration substance. The specific activity of the calibration substance is preferably known before the measurement. The calibration substance is preferably available for all investigated isotopes. Calibration is also in use one or more radioactive substances possible, which together spatially localize, contain known amounts of all isotopes to be measured. In this case, the specific activities or radiation intensities of each isotope in the calibration substance must be known. With the aid of suitable algorithms, it is possible on the basis of a plurality of local distributions determined to calculate the contribution of each isotope to the respective pixel value of the local distribution, one or more radiation intensities of the isotopes in the signal being changed in a local distribution. Obviously, in the latter case, other factors must be known, such as the differential level of radioactive decay, etc.

When an absorber is used, the change factors depend on the absorber material, its thickness and its mass absorption coefficient for the types of radioactive particles absorbed. If differential images are produced by decaying both isotopes, the change factors can be determined by measuring the relative intensities of the calibration substances for both isotopes. Or they can be calculated using information regarding the exact time and duration of each exposure and the decay rate constants for each radioisotope.

In a development of the invention, the ratio of the change factors of the quantity-specific radiation types is between 5% to 90%, preferably between 10% to 70%, in particular between 15% and 50%. The possibility of working with change factors that do not completely suppress a radiation type increases the number of radiation sources or detectors that can be used significantly. In particular when the change factors are implemented by radioactive decay of differently radiating isotopes, it is possible to calculate the separate spatial distributions of the isotopes even without complete decay of an isotope type. In a development of the invention, the change factor is implemented by using at least one absorber. An absorber reduces the intensity of the radiation (particles or photons) that passes through the material. The absorber can consist of metal, metal alloy, plastic, plexiglass, Teflon and / or another suitable material. For the function of the invention, it is essential that the emitted radiation types, for example photons from I-125 isoptopes and beta particles from I-131 isoptopes, which generate the signal on the detector, from the absorber with different change factors or Reduction factors are absorbed or damped. For this purpose, absorbers made of elements with a low atomic number are more effective, for example aluminum foil or thin plexiglass, since these absorbers absorb beta particles more than photons. Furthermore, absorbers made of a very thin layer of elements with a high atomic number can be used, since these absorbers absorb photons more than beta particles. The absorber thickness plays a key role in absorption efficiency.

In a development of the invention, the change factor is realized by radioactive decay of the respective point radiation objects of the at least two sets, the respective point radiation objects of the at least two sets preferably having different half-lives. The change or reduction in the radiation intensity here does not come about through an absorber, but rather through a difference in the decay rate of the point radiation objects. For example, isotopes 1-125 and 1-131 are well suited for this purpose because their half-lives differ by a factor of almost 7.5. However, other isotopes can also be used for the invention.

In a development of the invention, the change factor is determined by a different sensitivity of a detector used to determine a local distribution of the radiation intensity of the carrier surface realized for the respective quantity-specific radiation type. The change factors of the radiation intensities of the quantity-specific radiation types result here from the different sensitivities of the detectors used for the radiation emitted by the at least two quantities of point radiation objects. The different sensitivities are preferably chosen such that the ratio of the change factors is preferably greater than 5%.

In a further development of the invention, the spatial distributions of the radiation intensity are determined using a spatially resolving detector for alpha, beta, gamma and / or x-radiation.

In a development of the invention, the local distributions of the radiation intensity are determined using a so-called phosphor imager detector. Such a phosphor imager detector is also referred to as a phosphor imager plate or phosphor imager IP. The phosphor imager detector preferably works on the basis of photostimulated luminescence. Advantageously, a different sensitivity of a phosphor imager used for determining a local distribution of the radiation intensity of the carrier surface for the respective quantity-specific radiation type is achieved in that the phosphor of the phosphor imager additionally contains atoms with a high atomic number Z, in particular lead, which, for example, in of a lead-containing compound, and / or contains atoms with a low atomic number Z. If atoms with a high or low atomic number Z are introduced into the phosphor imager detector during the manufacture of the phosphor imager detector, its detection sensitivity for gamma radiation increases or decreases, for example. This means that the ratio of the sensitivities for different types of radiation changes. Additional atoms are built in that are not necessary for the storage of light in the phosphor imager detector, the absorption efficiency of the phosphor imager But change the detector. In this way, a distinction can be made between alpha, beta, gamma and neutron radiation and radiation of different energies. In particular, the ratio of the sensitivities for gamma radiation to that for beta radiation, in particular for beta radiation with energies greater than 20 keV, which can penetrate the phosphor jacket of the detector, can be changed:

1) Installation of elements with a high atomic number Z, 2) Installation of elements with a low atomic number Z.

In a further development, the first local distribution of the radiation intensity of the carrier surface is determined simultaneously by a first phosphor imager detector and the at least second local distribution of the radiation intensity of the carrier surface is determined by an at least second phosphor imager detector, the sensitivity of the first phosphor imager detector and the sensitivity of the at least second phosphor imager detector is different for the respective quantity-specific radiation type. This means that at least two phosphor imager detectors or phosphor imager IPs are used in the measurement, which have a different ratio of the sensitivities for the radiation of the different point radiation objects. This makes it possible to carry out the measurements or the determinations of the respective radiation intensities in quick succession or simultaneously. The different sensitivities of the different IP materials make it possible to calculate the spatial distribution of the respective point radiation objects, even if no sufficient difference has arisen due to the decay of the isotopes used. Of course, this procedure can also be combined with a waiting time between the measurements in order to additionally use a decay of the isotopes. In a development of the invention, the first and the at least second phosphor imager detector are attached to opposite sides of the carrier surface. Here, using phosphorimager IPs of different sensitivity, two measurements are carried out simultaneously or simultaneously on the front and back of the samples or carrier surfaces to be measured. The different sensitivity of the different IP materials makes it possible to calculate the local distribution of the point radiation objects.

In a further development of the invention, the local distributions of the radiation intensity are determined using a so-called flat detector. Such flat detectors or flat panel detectors enable the production of digital instant X-ray images. They convert X-rays directly into a matrix of digitally coded pixels, which increases the detection efficiency and the evaluation speed.

If the change factor is to be realized by a different sensitivity of a detector used to determine a local distribution of the radiation intensity of the carrier surface for the respective quantity-specific radiation type, a phosphor imager detector and a flat detector can be used, for example, for each measurement to carry out the spatial distribution of the radiation intensity of the support surface. The local distribution of the point radiation objects can be calculated individually from the local distributions measured with the different detectors.

In a development of the invention, the first and the at least second spatial distribution of the radiation intensity are determined using a similar detector. In principle, a detector does not allow clear discrimination between different types of radiation, for example beta particles or photons, which for example originate from two different isotopes, or radiation from isotopes with different half-lives. With the help of the above procedure, Using the same or a similar detector, a multi-color measurement is possible, ie a separate, overlay-free measurement of the spatial distribution of the individual types of point radiation sources. Identical detectors of the same type are detectors of the same type or of the same type, which for example do not differ or differ only insignificantly in their sensitivity to specific types of radiation. The ^ s simplifies the construction of an analysis device and thus saves costs.

In a development of the invention, at least two substances to be analyzed are each marked with a quantity of point radiation objects, the substances marked in this way are mixed and the mixture is then applied to the carrier surface, in particular in the form of a flat analysis means. The substances to be analyzed can advantageously be peptides, proteins and / or oligonucleotides, with, for example, a first and a second protein sample, which can each consist of a large number of different proteins, being labeled with different isotopes. After the labeling, the proteins of the two samples are mixed and applied together to an analysis means, in particular a protein gel, nucleic acid array, protein array, ELISA array and / or a blot. The substances or proteins of the different samples to be analyzed are then separated on the analysis means on the basis of specific properties which are determined, for example, by the analysis means. Following the separation, the local distribution of the point radiation objects for each set of point radiation objects is now calculated individually by the method according to the invention. The location distribution of the proteins to be examined of the respective samples can now be concluded separately from one another via the location distribution of the specific point radiation objects. Brief description of the drawings Advantageous embodiments of the invention described below are shown in the drawings, in which:

1A with a phosphor imager, local distribution of the radiation intensity of a mixture of 2 protein samples separated with the aid of a 2D gel, one sample being labeled with isotopes of type 1-125 and the other sample with isotopes of type 1-131,

1B with the phosphor imager, local distribution of the radiation intensity of the 2D gel from FL1A determined after a waiting time of 82 days,

1C, using the method according to the invention, calculated the spatial distribution of the radiation intensity of the protein sample which was labeled with isotopes of the type 1-125,

1 D using the method according to the invention, calculated the spatial distribution of the radiation intensity of the protein sample which was labeled with isotopes of the type 1-131,

Fig. 1E difference of the spatial distributions of Fig.1 C and Fig. 1 D, the protein sample which was labeled with isotopes of type 1-125 blue and the protein sample which was labeled with isotopes of type 1-131 orange is shown

1 F using the phosphor imager, the local distribution of the radiation intensity of the 2D gel from FIG. 1A determined after a waiting time of 23 days,

1 G using the method according to the invention calculated local distribution of the radiation intensity of the protein sample, which with I- sotopes of type 1-125 was marked, the local distribution from FIG. 1 F being used for the calculation as the second local distribution determined,

1 H using the method according to the invention, local distribution of the radiation intensity of the protein sample which was labeled with I- * sotopes of type 1-131, the local distribution of FIG. 1 F being used for the calculation as the second local distribution determined,

11 shows the difference in the spatial distributions of FIG. 1G and FIG. 1 H, the protein sample which has been labeled with isotopes of the type 1-125 being shown in blue and the protein sample which has been labeled with isotope of the type 1-131 in orange .

2A with a phosphor imager determined local distribution of the radiation intensity of protein samples from bovine serum albumin of different dilution levels and mixing ratios labeled with isotopes of the type 1-125 and isotopes of the type 1-131, without using an absorber,

2B with the phosphor imager determined local distribution of the radiation intensity of Fig. 2A using an absorber,

2C, using the method according to the invention, calculated the spatial distribution of the radiation intensity of the isotopes of type 1-125,

2D calculated with the aid of the method according to the invention, spatial distribution of the radiation intensity of the isotopes of type 1-131,

Fig. 2E difference of the spatial distributions of Fi.2C and Fig.2D, the isotopes of the type 1-125 blue and the isotopes of the type 1-131 range are shown. First embodiment

In a first embodiment of the invention, two protein samples, each consisting of a large number of different proteins, are labeled with different isotopes of the element iodine. The first sample is marked with 1-125, the second sample with 1-131. To calibrate the mass scale, special mass reference proteins are marked 1-125. 1-125 has a half-life of 60.14 days and decays due to electron capture, resulting in X-rays and gamma rays in the range from 27 keV to 35 keV. 1-131 has a half-life of 8.06 days and decays due to the various mechanisms of beta emission.

The samples are then mixed and separated using a 2D gel. To determine the change factors, calibration points for both isotope types are applied to the left side of the gel. They are used to calculate the change factors that are required when calculating the individual local distributions.

After the 2-dimensional separation, the phosphor imager IP is exposed to the sample or the radiation of the sample for a predetermined time. The Phosphorimager-IP is sensitive to low-energy X-rays, gamma rays and beta particles. The relatively high-energy photons of the 1-131 decay are absorbed or detected with a low efficiency by a phosphor imager IP. The phosphor imager IP integrates the radiation intensities of both radiation sources. The spatial distribution of the stored radiation energy of the phosphor imager IP is then read off with a reading device and imaged in a pixel matrix or an image, which is shown in FIG. 1A. The local distribution or the pixel matrix determined in this way contains portions that come from both 1-125 and 1-131. After a waiting period of 82 days, the local distribution of the sample is ben described again determined. This local distribution is shown in Fig. 1B. Due to the different half-lives of the two isotopes, the radiation intensity or the activity of 1-125 has decreased by a factor of approximately two. In contrast, the radiation intensity of the 1-131 remaining on the gel is less than 1% of the original radiation intensity. In Fig. 1 B there are therefore only signal components from 1-125.

In this embodiment, the different change in the radiation intensities is brought about exclusively by the inherent properties of the different radioactive decay of the two isotopes, but not by an absorber.

The spatial distribution of the individual isotopes is then calculated separately using the method according to the invention. For the calculation, the intensity over a range of 4X4 neighboring pixels was used or summed, using a Gaussian filter. The determined local distributions are aligned with one another on the basis of corresponding reference points, which are present at the same locations in the local distribution. 1C shows the calculated local distribution of the radiation intensity or the isotope amount of the protein sample which was labeled with isotopes of type 1-125 and FIG. 1D shows the calculated local distribution of the radiation intensity of the protein sample which was labeled with isotopes of type 1-131 , FIG. 1E shows the difference in the spatial distributions of FIG. 1C and FIG. 1D, the protein sample which was labeled with isotopes of type 1-125 being blue and the protein sample which was labeled with isotopes of type 1-131 , are shown in orange. Black means that there is an equal number of both isotopes.

The local distribution of the radiation intensity can of course also be determined without a spatially resolving detector. For this purpose, the carrier surface to be examined is separated by suitable wise cutting, cut into smaller pieces and the radiation intensity of these pieces determined by a conventional "single-source" detector. The radiation type and the radiation intensity can be determined, for example, using gamma and / or beta spectroscopy.

Second embodiment

In a second embodiment of the invention, with the same procedure as in the first embodiment, the waiting time between the determination of the first and the second local distribution is reduced from 82 days to 23 days. 1 F shows the local distribution of the radiation intensity of the 2D gel from FIG. 1A determined with the phosphor imager after a waiting time of 23 days. Due to the incomplete decay of 1-131, both isotopes now contribute to the determined spatial distribution. The change factors for both isotopes are therefore significantly greater than 0%. Likewise, the ratio of change factors is much greater than 0%.

1G shows the local distribution of the radiation intensity of the protein sample calculated with the aid of the method according to the invention, which was labeled with isotopes of the type 1-125, FIG. 1 H shows the local distribution of the radiation intensity of the protein sample calculated analogously, that with isotopes of type 1 -131 was marked, the local distribution of FIG. 1 F being used for the calculation in both cases as the second determined local distribution.

FIG. 11 shows the difference in the spatial distributions of FIG. 1G and FIG. 1 H, the protein sample which was labeled with isotopes of type 1-125 being blue and the protein sample which was labeled with isotope of type 1-131 orange is shown. Black means that there is an equal number of both isotopes. The results of this embodiment coincide with the results of embodiment 1, which can serve as reference results due to the almost complete suppression of 1-131. The method according to the invention consequently delivers reliable measurement results even if a radiation type is not completely suppressed when determining the second local distribution.

Third embodiment

In this embodiment of the invention, an absorber is used to implement the corresponding change factors, i.e. to make differential measurements of the sample containing the two isotopes 1-125 and 1-131. Analogously to the first embodiment of the invention, a two-dimensional gel is produced which contains the proteins labeled with the two isotopes 1-125 and 1-131. The phosphor imager IP is exposed to the spatial distribution of the radiation intensity of the 2D gel for a predetermined time. The phosphor imager IP is then read by the phosphor imager reader. The radiation intensity over the area of the 2D gel is depicted in a matrix, the values of the individual elements of the matrix or of the pixels representing the radiation intensity of the assigned area on the phosphor imager IP. A matrix or image is formed, the signal components of which come from both 1-125 and 1-131.

In order to separate the signal components, it is necessary to change or reduce the intensity of a radiation type by suitable means and to determine a second image or a second spatial distribution.

By introducing an absorber of known thickness between the sample and the phosphor imager IP during the irradiation, the radiation intensity can be selectively for each radiation type with different Change factors are reduced. Such an absorber can consist of metal, metal alloy, plastic, plexiglass, Teflon or another suitable material. In order to achieve the function according to the invention, the absorber must in each case have different change factors for the radiation type emitted by the radiation sources (in this embodiment photons from 1-125 and beta particles from 1-131), which generates the signal on the detector absorb.

In this embodiment, the intensity of the signal originating from 1-131 relative to the signal from 1-125 can be significantly reduced with the aid of the absorber. For this purpose, absorbers made of elements with a low atomic number are more effective (example: aluminum foil, thin plexiglass, etc.). The absorber thickness plays a key role in absorption efficiency.

The first and the at least second local distribution of the radiation intensity can be determined simultaneously using a "sandwich-like" arrangement of phosphor imager IP, gel, absorber, phosphor imager IP. Alternatively, the images could be taken in succession, with or without absorber, which is preferred in the invention. Obviously, other radioactive isotopes can also be used for the invention disclosed herein.

Fourth embodiment

In a fourth embodiment of the invention, as in the third embodiment, an absorber is used in order to change the radiation intensity of the different radiation types differently.

In this embodiment, two samples of bovine serum albumin are labeled once with 1-125 and once with 1-131. The samples are diluted four times in 10 steps. So it arises from everyone Sample 5 dilution levels each, from undiluted to 1/10000 diluted. The radiation intensities therefore differ by a factor of 10000. Each dilution level of the first sample is mixed with each dilution level of the second sample. This results in a total of 25 samples with different mixing ratios, each of which is applied in a volume of 1 μl to a square filter paper with a side length of 3 nm. The filter papers are arranged in a matrix arrangement on a flat carrier. The concentration of 1-125 decreases from bottom to top. The concentration from 1-131 decreases from left to right.

A phosphor imager IP is exposed to the radiation from the flat carrier for 24 hours. The phosphor imager IP integrates the radiation intensities of both radiation sources of the samples. The spatial distribution of the stored radiation energy of the phosphor imager IP is then read using a reading device and imaged in a pixel matrix or an image, which is shown in FIG. 2A. The local distribution or the pixel matrix determined in this way contains portions that come from both 1-125 and 1-131. Immediately after this first local distribution has been determined, a second local distribution is determined, an absorber being arranged between the flat support and the phosphor imager IP. This second spatial distribution is shown in Fig. 2B. The absorber consists of a 900 μm thick plastic layer. The radiation intensities of the samples arranged on the flat carrier were integrated or summed up for both local distributions determined using the same areas. On the basis of these integrated radiation intensities, the local distribution of the individual isotopes is calculated separately from the two local distributions determined using the method according to the invention. FIG. 2C shows the calculated local distribution of the radiation intensity or the isotope quantity of the protein samples which were labeled with isotopes of the type 1-125 and FIG. 2D shows the calculated local distribution of the radiation intensity of the protein samples which were labeled with isotopes of the type 1-131. Figure 2E shows the difference of the local distributions of FIG. 2C and FIG. 2D, with the protein samples labeled with isotopes of type 1-125 being shown in blue and the protein samples labeled with isotopes of type 1-131 being shown in orange. Black means that there is an equal number of both isotopes.

The undiluted sample labeled 1-125 in the lower right and the undiluted sample labeled 1-131 in the upper left were used to calculate the decay constants required to calculate the spatial distributions.

The calculated result agrees with the theoretically expected result within narrow error limits. This shows that the method according to the invention also works with change factors that are clearly above 0%.

Possible variations of the embodiments

In the above embodiments of the invention, analyte molecules (proteins) are labeled with chemically identical radioisotopes (radioactive iodine). This method is extremely useful for performing differential analyzes because the differences in the intensity distribution of the radiation between the two samples are due to differences between the samples themselves. This also applies to proteins labeled with P-33 and P-32, for example.

With a combination of proteins labeled with C-14, S-35 or P-33, different 2D PAGE images are generated because carbon, sulfur and phosphate are present in the proteins in different chemistries. For example, some proteins have no cysteine or methionine and therefore do not carry sulfur, and are nevertheless phosphorylated by a kinase so that they carry phosphate efficiently. Other proteins can have many sulfur-containing amino acids and yet not be phosphorylated. Nevertheless, the present invention is also suitable for extracting the synthetic images which correspond to the distribution of both isotopes when a combination of these isotopes is used according to the invention. In fact, the invention is applicable to the differential measurement of any radioisotope that is distributed on a surface or a similar surface, even those with different surface structures that are suitable for phosphor imager analysis.

The radioactive isotopes suitable for the invention have significant differences in the decay rate, the energy emitted particles, the type of the emitted particles or a combination thereof. High isotope purity is desirable but not necessary.

A high chemical purity of the radioisotopes is desirable, but not necessary. It is possible to apply the invention to samples labeled with various mixtures of isotopes, e.g. labeling one sample with 95% 1-131, 5% 1-125 and labeling the other sample with 5% 1-131, 95% 1-125. The labeling mixture can obviously be quite complex without departing from the idea or scope of the invention and could, for example, contain trace amounts of many isotopes, some of which are not covalently incorporated into the analyte molecules, such as K-40.

The present invention is independent of the method of introducing radioactive isotopes into the analyte molecules. For example, proteins can be labeled by metabolic uptake, by radioiodination posthan / est, by alkylation with radioactive reagents (any chemistry) or by other methods.

Another application of phosphor imagers is the measurement of DNA and nucleic acid arrays. In such applications as the frequent analysis of mRNA, P-33, P-32, S-35, C-14, H-3 and other isotopes can all be introduced into hybridization molecules in a conventional manner. By the principles of the invention, a phosphor imager can be used to perform two-color analyzes of these systems. Because of the simple chemistry of DNA-like polymers, the radiation intensities of hybridization molecules containing these radioisotopes can all be identical if the same sample is independently labeled and measured for each isotope. The principles described above obviously also apply to protein arrays or all other arrays or semi-planar distributions of analyte molecules which are suitable for phosphorimager analysis.

It will be appreciated that applications of the invention can be implemented in the field of medical imaging where two or more differential images are created to extract synthetic images from two or more signal sources. An example could relate to the detection of certain radioactive trace elements combined with X-rays of the body using suitable absorbers according to the invention, and could be of great advantage in the field of medicine.

Obviously, the present invention is not limited to phosphor imager IP or radioactive measurements. The invention can also be applied to cases in which visible light and not radioactivity are measured several times and both signal components are extracted algorithmically. For example, an enzyme reaction can produce light of a similar wavelength to that emitted by a fluorophoric or luminescent molecule. By determining the intensity distribution of the light emitted jointly by the chemical reaction and the fluorophore and the intensity distribution of the light emitted by the fluorophore after the enzymatic reaction produces a reduced amount of light, the contributions of the fluorescent and enzymatically produced light can be determined in the original image. With this implementation it is important It is important that the reduction in the intensity of both light sources is known or that it can be precisely calibrated.

All publications and patent applications cited in this disclosure are incorporated by reference into the disclosure of the invention.

Terminology, material and methods

The terminology used is explained below and an overview of the conventional materials and methods is given.

An image is represented by a matrix, the matrix cell values corresponding, for example, to the intensity of a signal that is detected by a detector at a corresponding location on the imaged surface. The images can be saved in the usual file formats for images.

A multicolor analysis is understood to mean that several samples are marked, mixed and separated separately. The evaluation is then carried out individually for each sample, unaffected by the existence of the other samples. There are several ways to generate multicolor equations.

Genomics means the quantitative investigation of nucleic acids or polymers similar to nucleic acids.

Proteomics was defined as the study of all proteins expressed by the genomes of the cells examined. The acronym PROTEOM means PROTEins that are expressed by a GenOM. Wasinger VC, Cordwell SJ, Cerpa-Poljak A, Yan JX, Gooley AA, Wilkins MR, Duncan MW, Harris R, Williams KL, Humphery-Smith I. (1995) Progress with gene-product mapping of the Mollicutes: Mycoplasma genital. Electrophoresis, 16, 1090-4.

Here proteomics specifically means the study of molecules in which at least part of it has been translated into a ribosome (a protein), as is known to those skilled in the art, or a group of such proteins. Proteomics also includes the study of post-translational protein modifications, protein synthesis and degradation rates, protein degradation products and combinations thereof.

Here, the molecular profile or profile of a biological system, such as a cell, a group of cells, a tissue or an organism, refers to a pattern of changes in gene expression or protein expression or lipid composition or metabolite production by small molecules or temperature or metabolite secretion by small ones Molecules, changes in sugar frequency or types, or changes in post-translational protein modifications, or changes in protein proteolysis, or changes in ion secretion by one or more cell compartments, or in ion uptake by one or more cell compartments between two or more examined biological systems. More generally, the molecular profile of a biological system is a measure of the atoms that make up that system, and especially their chemical, spatial, and temporal relationships.

Array means an orderly placement or arrangement. The term is used here to refer to an orderly placement of oligonucleides (including RNA, cDNA and genomic DNA) or ligands for analyte molecules such as affinity reagents for proteins, lipids and sugars or molecules containing such functional groups (e.g. contains a Glycoprotein at least one sugar group or e.g. holds a lipoprotein to designate at least one lipid group). The arranged molecules are positioned on a surface, for example a chip, and are used to capture complementary oligonucleotides (including RNA, cDNA and genomic DNA) or substrates for the ligand. Because the oligonucleotide or ligand is known in each position in the array, the sequence (eirfer nucleic acid) or a physical property (of a protein) can be determined by the position at which the nucleic acid or substrate binds to the array.

Protein separation methods

The use of electrophoresis for preparative purposes is an established technique and there are different types of electrophoresis devices for preparative and analytical purposes. These devices and their accompanying principles can be divided into three categories.

Andrews AT. (1986) Electrophoresis: Theory, Techniques, and Bio-chemical and Clinical Applications. Oxford University Press.

Westermeier R. (1977) Electrophoresis in Practice: A Guide to Methods and Applications of DNA and Protein Separations. John Wiley and Sons, Weinheim.

a) Zone electrophoresis b) Isotachophoresis c) Isoelectric focusing

Isotachophoresis uses hydrophilic matrices and typically shows high resolution but low loading capacities. In combination with free electrophoresis, the method can be used for micro-preparative purposes. Weber G, Bocek P. (1998) Stability of continuous flow electrophoresis. Electrophoresis, 19, 3094-3905.

Isoelectric focusing (IEF, isoelectric focusing) is carried out either in liquid density gradients, in gel gradients or in multi-chamber devices, with a separation gel medium based on IEF immobilin.

Righetti PG. (1990) Immobilized pH gradients: theory and methodology. In: Laboratory techniques in biochemistry and molecular biology. Vol. 20 (ed. RH Burdon, PH Knippenberg). P. 397. Elsevier, Amsterdam. Righetti PG, Bossi A, Wenisch E, Orsini G. (1997) Protein purification in multicomponent electrolyzers with isoelectric membranes. J. Chromatog. B Biomed. Be. Appl., 699, 105-15.

Zone electrophoresis comprises glycine, bicin and tricin buffered SDS-PAGE (sodium dodecylsulfate poyacrylamide gel electrophoresis) protein systems, which are commonly used in protein analysis and are discussed in the literature references mentioned above.

Current proteomics techniques usually involve the use of 2D-PAGE (two-dimensional polyacrylamide gel electrophoresis), whereby proteins are first separated by differences in the pl by IEF in the first dimension. These proteins are then separated by the molecular weight in an SDS-PAGE gel in the second dimension.

While 2D-PAGE is currently the method that provides the highest separation performance for biochemical cleaning, the number of proteins and their modified isoforms found in eukaryotic cells, tissues and organisms exceeds the separation capacity of the best 2D-PAGE system , The abundance of different proteins and protein isoforms in complex eukaryotic cells, tissues and orga- nisms also fluctuates strongly. The most common proteins in a eukaryotic cell can exist with more than 10 ⁸ copies per cell. Examples of common proteins include certain cytoskeletal proteins or albumin in the liver and blood serum. Some proteins can have significant biological effects when they are present in very small amounts, perhaps with only a few molecules per cell. Although the most limited amount at which proteins can exert biological activity is poorly understood, this category could include examples such as telomerase and DNA polymerase in the progression from precancerous to cancerous cells. Other cases when low frequency molecules are biologically relevant in protein mixtures are in the blood where a few molecules of cytokine per liter can be important or bone marrow where only one metastatic cancer cell among trillions of other cells can have fatal biological consequences for the patient. For a discussion of the number of proteins and protein isoforms to be expected in eukaryotic cells and some implications for 2D PAGE in proteomics, see Vuong et al.

Vuong GL, Weiss SM, Kammer W, Priemer M, Vingron M, Nordheim A, Cahill MA. Improved sensitivity proteomics by post harvest alkylation and radioactive labeling of proteins. 2000, Electrophoresis 21: 2594-2605.

The reproducibility of 2D-PAGE is also notoriously low, so that typically several replicates of each gel are required in order to use software packages to put together an average approximation of the composition of the typical 2D-PAGE pattern of the examined system. Some software packages are commercially available. An important application of proteomic studies is to determine the difference in the frequency of certain proteins or protein isoforms between two or more experimentally interesting regimes, such as proteins from control cells, tissues or organisms, compared to corresponding frequencies from pharmacologically treated cells, tissues or organisms.

A number of other methods are available for protein separation, including but not limited to high pressure liquid chromatography, thin layer chromatography, FPLC, gel filtration chromatography, ion exchange chromatography, microfluidic systems such as capillary electrophoresis and chip-based microfluidics, affinity reagents with microaffinity arrays and electrophoresis. The following references describe the state of the art of separation processes that are used in proteomics research.

Daniel C. Liebler. Introduction to Proteomics: Tools for the New Biology. 300 pages 1st edition (Nov. 2001) Humana Press; ISBN: 0896039927. Timothy Palzkill. Proteomics. 1st ed. (Nov. 15, 2001) Kluwer Academic Publishers; ISBN 0792375653.

MJ Dünn (ed.) Proteomics Reviews 2001. (April 2001) VCH Verlagsges. mbH; ISBN: 3527303146. Andre Schrattenholz (ed.). Methods of proteome research. Molecular analysis of protein expression. 2001. Spectrum Academic Publishing House, Heidelberg. ISBN: 382741 153X.

Issaq HJ. The role of separation science in proteomics research. Electrophoresis 2001, 22: 3629-3638. Figeys D, Pinto D. Proteomics on a Chip: promising developments. Electrophoresis 2001, 22: 208-16.

Blagoev B, Pandey A. Microarrays go live - new prospects for proteomics. Trends biochem. Be. 2001, 26: 639-641.

Cahill DJ. Protein and antibody arrays and their medical applications. J Immunol Methods 2001, 250: 81-91. Paweletz CP, Charboneau L, Bichsel VE, Simone NL, Chen T, Gillespie JW, Emmert-Buck MR, Roth MJ, Petricoin III EF, Liotta LA. Reverse phase protein microarrays which capture disease progression show acti- vation of pro-survival pathways at the cancer invasion front. Oneogene 2001, 20: 1981-1989.

Figeys D, Pinto D. Proteomics on a chip: promising developments. Electrophoresis 2001, 22: 208-16. Miklos GL, Maleszka R. Protein functions and biological contexts. Proteomics 200, 1: 169-178.

LorHsh H, Berk A, Zipursky LS, Matsudaira P, Baltimore D, Darneil J. Molecular Cell Biology, 4th ed. WH Freeman, New York, ISBN: 0-7167-3136-31986. Patterson SD. Proteomics: the industrialization of protein chemistry. Curr. Opin. Biotechnol. 2000 11: 413-418.

Protein detection in proteomics

Proteins can be detected by a variety of methods described in the references above, which include, but are not limited to:

* 1) Combination of dyes or staining agents such as amid black, sulforhodamine B, agalm black or various silver staining processes (literature references above). 2) Spectroscopic detection of aromatic amino acids or

Peptide bonds.

3) Non-covalent association of fluorescent reagents.

4) Covalent binding of fluorescent reagents, such as monobromobimanthiolyt, naphthalene-2,7-disulfonic acid, fluorescene, Cye dye derivatives or a number of other reagents, which the

Are known to experts. The introduction of fluorescent groups in protein molecules can e.g. B. by alkylation of amino groups such as lysine or the amino end group, by alkylation of cysteine groups or by other methods.

5) Metabolic introduction of radioactive isotopes including H-3, C-14, S-35, P-32 and P-33 followed by radioactive detection.

6) Postharvest radioactive labeling, such as protein iodination (Vuong et al, see citations above and there), followed by radioactive detection.

7) Immunodetection where antibodies or recombinantly produced affinity molecules specifically interact with proteins of interest, following a series of standard protocols such as those found in Harlow and Lane, Antibodies, A Laboratory Manual (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1988) be taught or as taught by Hudson and Souriau, Expert Opin. Biol. Ther. 1: 845-855 (2001) and Manoutcharian et al., Curr. Pharm. Biotechnol. 2: 217-223 (2001). antibody can be radiolabeled with radioactive molecules or bound covalently, which allows their detection with radioactive detectors, as described by Manoutcharian et al. is described above. 8) Tagging is another way of detecting and determining changes in protein expression. For example, the gene encoding the protein can be engineered to produce a hybrid protein that contains a detectable label (tag) so that the protein can be specifically detected by recognizing the label. Systems are available that allow direct imaging and quantitative analysis of radioactive labels, e.g. B. in gels on which proteins have been separated. Differences in expression can be determined by recognizing differences in the amount of markings present in test and control samples.

In cases where sample material is extremely limited, such as certain biopsy or preservation methods for obtaining tissue from patients in clinical situations, or where the examination should include detection of the analyte molecules with the least frequency, the most sensitive detection methods are highly desirable. Of the above-mentioned methods, the detection of radioisotope-labeled proteins by suitable detectors is several orders of magnitude more sensitive than any other method and is therefore the means of choice.

Image Analysis Software

There are some complex software packages for processing graphic or digital data obtained from biological studies, such as protein gel analysis or nucleic acid array analysis. examples Games for proteome analysis include the products Phoretix 2C from Non-Linear Dynamics (Newcastle upon Tyne, UK), Delta 2D from Decodon GmbH (Greifswald, Germany), 3Z from Compugen (Tel-Aviv, Israel), Gellab 11+ from Scanalytics (Faifax , VA), Bioimage from Genomic Solutions (Ann Arbor, MI), Melanie 3 from GeneBio (Geneva), the Keppler program from Large Scale Biology Corporation (Vacaville, CA), the Java browser of the CAROL program (Free University , Berlin), the gel image comparator Flicker 2D gel image comparator (Lemkin PF. Comparing two-dimensional electrophoretic gel images across the Internet. Electrophoresis, 18: 461-470 (1997) and image analysis products by Amersham Biosciences (Freiburg, Germany) sold.

Protein identification in proteomics

Mass spectrometry is becoming increasingly important in proteomics, not only for the identification of proteins separated by 2D-PAGE, but also for the direct detection and relative quantification of proteins independent of 2D-PAGE. Other methods of protein identification include Edman degradation, amino acid analysis, and other methods known to those skilled in the art. Many of these methods involve collecting data from the protein under study and comparing these parameters to a list of parameters predicted by theoretical analysis of nucleic acid and protein databases.

Anderson NL, Matheson AD, Steiner S. Proteomics: applications in basic and applied biology. Curr. Opin. Bioltechnol. 2000, 11: 408-412. Gygi SP, Aebersold R. Mass spectrometry and proteomics. Curr. Opin. Chem. Biol. 2000, 4: 489-494.

Lee KH. Proteomics: a technology-driven and technology-limited discourse science. Trends in biotechnology. 2001 Jun; 19 (6): 217-222. Patterson SD. Proteomics: the industrialization of protein chemistry.

Curr. Opin. Biotechnol 2000, 11: 413-418.

Godovac-Zimmermann J, Brown LR. Perspectives for mass spectrometry try and functional proteomics. Mass Spectrom. Rev. 2001, 2001, 20: 1-

57th

P. James (ed.) Proteome Research: Mass Spectrometry (Principles and * Practice) 235 pages (Dec. 2000) Springer Verlag; ISBN:

3540672567th

genomics

A number of methods for detecting and comparing the expression of gene expression in cells, organisms or viruses or other transcription-active systems are known in the prior art.

A standard method for such comparisons is the Northern blot. In this technique, RNA is extracted from the sample and applied to a series of gels suitable for RNA analysis, which are then used to separate the RNA according to size.

Sambrook, J. et al., 1989. Molecular Cloning, A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 2nd ed.

The gels are then blotted (as described above for Sambrook) and hybridized to samples for RNAs of interest. The samples can be radioactive or non-radioactive. For example, hybridization with the sample can be viewed and analyzed by chemiluminescence detection of the bound samples with the Genius system (Boehringer Mannheim Corporation, Mannheim, Germany) according to the manufacturer's instructions. An equal loading of the RNA in the webs can be achieved, for example, by staining the ribosome RNA Bands assessed with ethidium bromide. Alternatively, the samples can be radiolabeled and detected autoradiographically with a sample for a gene and a photographic film or a phosphor imager or according to the present invention with samples for more than one gene.

The RNA can be amplified by a number of methods and then detected. For example, Marshall, US Patent No. 5,686,272 discloses the amplification of RNA sequences with the ligase chain reaction LCR (ligase chain reaction). LCR is from Landegren et al., Science, 241: 1077-1080 (1988); Wu et al., Genomics, 4: 560-569 (1989); Barany, in PCR Methods and Application, 1: 5-16 (1991); and Barany, Proc. Natl. Acad. Be. USA, 88: 189-193 (1991). The RNA can also be converted into complementary DNA (cDNA) by reverse transcription and then amplified by LCR, polymerase chain reaction (PCR, polymerase chain reaction) or other methods. An example of a method for performing reverse transcription of RNA is disclosed in U.S. Patent No. 5,705,365. Selection of suitable primers and PCR protocols is taught, for example by Innis, M. et al., Ed. PCR Protocols 1990 (Academic Press, San Diego CA). Differential expression of messenger RNA (mRNA) can also be compared by reverse transcription of mRNA into cDNA, which is then cleaved by restriction enzymes and separated electrophoretically, so that a comparison of the cDNA fragments is possible, as is the case with Belavsky, U.S. Patent No. 5,814,445.

Typically, primers are labeled at the 5 'end with biotin or one of the many fluorescent dyes. Samples are usually labeled with an enzyme, such as radish peroxidase (HRP, horse radical peroxidase) and alkaline phosphatase, see Levenson and Chang, Nonisotropically Labeled Probes and Primers in: Innis et al. B. marked with biotin psoralen. Detailed sample protocols for labeling primers and synthesizing enzyme-labeled samples are given by Levenson and Chang. The samples can also be labeled with radioactive isotopes. An exemplary protocol for synthesizing radioactively labeled DNA and RNA samples is carried out in Sambrook. A number of reagents are available for introducing various radionuclides into DNA samples, including enzyme precursor substrates with 3H, 14C, 32P, 35S and other elements that are not present in naturally occurring nucleic acids, such as. B. radioactive iodine. Furthermore, primers can be made that contain a wide range of other radioactive elements that are covalently bound to the primer, as is known to those skilled in the art. A number of methods for the detection of PCR products are known. Generally, a step is included that allows hybridization of the sample and the PCR product, followed by one or more development steps to enable detection.

If a radioactively labeled sample is to be used, PCR products with which the sample is hybridized can be detected by autoradiography. As an example, biotinylated dUTP (Bethesda Research Laboratories, MD) can be used in amplification. The labeled PCR products can then be separated on an agarose, placed on a nylon filter by Southern transfer and detected, for example, by a detector system with streptavidin / alkaline phosphatase. A protocol for the detection of introduced biotinylated dUTP is e.g. B. in Lo et al., Ineorporation of Biotinylated dUTP, in: Innis et al. described. Finally, the PCR products can be applied to agarose gels and nucleic acids can be detected by a dye, such as ethidium bromide, which specifically recognizes nucleic acids.

Sutcliffe, U.S. Patent No. 5,807,680 teaches a method for simultaneously identifying differentially expressed mRNAs and measuring relative concentrations. The technology that supports the formation of cDNA with anchor encompassed by PCR followed by the visualization of almost all mRNA expressed in a tissue as a clear band on a gel, the intensity of which roughly corresponds to the concentration of mRNA.

Another group of techniques used to analyze the relative Trä ^* τιskπptexpressionswerte. Recently, four such approaches have been developed to enable comprehensive, high-throughput analysis. First, cDNA can be obtained by reverse transcription from RNA in the sample (as described in the above citations) and subjected to simple sequencing of the 5 'and 3' ends to generate expressed sequence markers (EST, expressed sequence tags) for the to define genes and control samples expressed in the test. Enumerating the relative representation of the markers from different samples gives an estimate of the relative representation of the gene transcript in the samples.

Second, a variation of ESTs, known as SAGE (serial analysis of gene expression), has been developed that enables quantitative and simultaneous analysis of a large number of transcripts. The technique uses isolation of short diagnostic sequence markers and sequencing to reveal patterns of gene expression characteristics of a target function, and has been used to compare expression values, e.g. B. of thousands of genes in normal and tumorous cells. See Veleulescu, et al., Science 270: 368-369 (1995), Zhang et al., Science 276: 1268-1272 (1997).

Third, approaches based on the differential display were developed. In these approaches, fragments defined by specific sequence delimiters can be used as the only identifiers for genes if they are coupled with information about the fragment length in the expressed gene. The relative representation of an expressed gene in a cell can then be determined by the relative representation tion of the fragment assigned to the gene can be estimated. Examples of some approaches are restriction enzyme analysis of differentially expressed sequences (READS) used by Gene Logic Inc., and total gene expression analysis by Digital Gene Technologies Inc. CLONTECH, Inc. ( Palo Alto, CA) distribute the Defta® Differential Display Kit for the identification of differentially expressed genes by PCR. Another method is the GeneCalling system from CuraGen Corp., New Haven CT, which combines gene identification with a database query of a restriction endonuclease impression, confirmed by comparative PCR using gene-specific oligonucleotides, which optimizes gene isolation procedures.

Fourth, in preferred embodiments, detection is performed by one of a number of hybridization analysis techniques. In these approaches, RNA from the sample of interest is subjected to reverse transcription to obtain encoded cDNA. The cDNA is then hybridized, typically with oligonucleotides or cDNAs of known sequence, which are arranged on a chip or another surface in a known sequence. The location of the oligonucleotide that hybridizes with the labeled cDNA gives sequence information about the cDNA, while the amount of labeled RNA or cDNA gives an estimate of the amount of relative RNA or cDNA in the original sample. Furthermore, the technique enables simultaneous hybridization with two or more different detectable markers, such as two or more radioactive or fluorescent markers, according to claim 1 of the present invention. The hybridization results provide a direct comparison of the relative expression of the samples. The prior art is given as an overview by King and Sinha, JAMA 286: 2280-2208 (2001) and Jain, Science, 294: 621-623 (2001) and their literature references. A number of hybridization analysis kits are commercially available. These kits enable the identification of specific RNA or cDNA molecules on high-density formats, including filters, microscopic plates, microchips and techniques of mass spectrometry. For example, Affymetrix, Inc. (Santa Clara, CA) distributes GeneChip® probe arrays that contain thousands of different oligonucleotide samples of known sequences, lengths, and locations in the array for high-precision gene sequencing. Synthetic oligonucleotide arrays combined with PCR methods can detect transcription ratios with one copy per cell in complex biological samples, and an ink jet oligonucleotide array technique can be used for precise application. See Hughes, TR, et al., Nature Biotechnology, 19: 342-347 (2001). CLONTECH's Atlas® cDNA expression array enables expression patterns of 588 selected genes to be measured. The Gene Discovery module from Hyseq Inc. (Sunnyvale, CA) enables screening of RNA with high throughput, without previous sequence information and a sensitivity of 1 mRNA copy per cell. Incyte Pharmaceuticals Inc. (Palo Alto, CA) offers microarrays that contain, for example, ordered oligonucleotides from human cancer and signal transduction genes. Techniques used by other companies are e.g. B. in Service, R .; Science 282: 396-399 (1998) and in Clarke PA Biochem. Pharmacol., 62: 1311-1136 and their citations.

Measurement of radioactive radiation

The first electrical devices that were developed to detect radiation were ionization detectors. These instruments are based on the direct capture of electrons and ions that are generated in a gas by radiation. There are two basic types of detectors known as ionization chambers and proportional counters.

Another detection device is the scintillation detector. It is based on the fact that certain materials, when struck by a nuclear particle or by radiation, have one or more photons, i. H. emit a scintillation. When coupled to an amplification device, such as a photomultiplier, these scintillations can be converted into electrical pulses, which can then be analyzed and electronically counted to provide information about the incident radiation.

Semiconductor detectors are also widely used detection devices based on crystalline semiconductor materials, especially silicon and germanium. These detectors are also called solid detectors. The principle of operation of semiconductors is analogous to that of gas ionization devices. Instead of a gas, the medium is a solid semiconductor material. The advantage of semiconductor detectors compared to ionization chambers is their greater energy resolution.

The following textbook describes the principles of radioactive detection methods:

Knoll, G.F .: Radiation detection and measurements, 802 S., 3rd ed. December 1999, John Wiley and Sons, ISBN 0471073385.

Claims

claims

1.Method for determining the local distribution of at least two sets of point radiation objects, each with a quantity-specific radiation type, on a common carrier surface, with the following steps: a) * determining a first local distribution of the radiation intensity of the carrier surface, b) changing the intensity of at least one quantity-specific radiation type with an associated one Change factor, c) determining at least one second local distribution of the radiation intensity of the carrier surface and d) calculating the local distributions of each of the at least two sets of point radiation objects individually from the first and the at least second determined local distribution.

2. The method according to claim 1, characterized in that the change step b) and step c) for determining the at least second spatial distribution of the radiation intensity of the carrier surface, preferably with a different change factor, are repeated at least once.

3. The method according to claim 1 or 2, characterized in that a determined local distribution of the radiation intensity is represented by an associated pixel matrix, wherein a pixel value of the matrix represents the radiation intensity of an associated location on the support surface.

4. The method according to claim 3, characterized in that the spatial distribution of each of the at least two sets of point radiation objects is determined individually on the basis of the pixel matrices.

5. The method according to claim 3 or 4, characterized in that the spatial distribution of each of the at least two sets of point radiation objects is determined individually on the basis of intensities that result from the summation of pixel values of defined, in particular adjacent, elements of the pixel matrix.

6. The method according to any one of the preceding claims, characterized in that each set of point radiation objects consists of at least one radiating, in particular radioactive, type of isotopes.

7. The method according to any one of the preceding claims, characterized in that each set of point radiation objects consists of light-emitting, in particular fluorescent, phosphorescent and / or luminescent, substances.

8. The method according to any one of the preceding claims, characterized in that there is at least one calibration point with known radiation for at least one set of different point radiation objects at at least one location on the support surface.

9. The method according to any one of the preceding claims, characterized in that the ratio of the change factors of the quantity-specific radiation types is between 5% to 90%, preferably between 10% to 70%, in particular between 15% and 50%.

10. The method according to any one of the preceding claims, characterized in that the change factor is realized by using at least one absorber.

11. The method according to any one of the preceding claims, characterized in that the change factor is realized by radioactive decay of the point radiation objects of the at least two sets, the respective point radiation objects of the at least two sets preferably having different half-lives.

12. The method according to any one of the preceding claims, characterized in that the change factor is realized by a different sensitivity of a detector used to determine a local distribution of the radiation intensity of the carrier surface for the respective quantity-specific radiation type of the at least two sets of point radiation objects.

13. The method according to any one of the preceding claims, characterized in that the local distributions are determined with a spatially resolving detector for alpha, beta, gamma and / or x-rays.

14. The method according to any one of the preceding claims, characterized in that the local distributions are determined with a so-called phosphor imager detector.

15. The method according to claim 14, characterized in that a different sensitivity of a phosphor imager detector used for determining a local distribution of the radiation intensity of the carrier surface is achieved for the respective quantity-specific radiation type in that the phosphor of the phosphor imager detector additionally contains atoms contains a high atomic number Z, in particular lead, and / or atoms with a low atomic number Z.

16. The method according to claim 15, characterized in that the first spatial distribution of the radiation intensity of the support surface by a NEN first phosphor imager detector and the at least second local distribution of the radiation intensity of the support surface are determined simultaneously by an at least second phosphor imager detector, the sensitivity of the first phosphor imager detector and the sensitivity of the at least second phosphor imager detector for each quantity-specific radiation type is different.

17. The method according to claim 16, characterized in that the first and the at least second phosphor imager detector are attached to opposite sides of the support surface.

18. The method according to any one of the preceding claims, characterized in that the local distributions are determined with a so-called flat detector.

19. The method according to any one of the preceding claims, characterized in that the first and the at least second spatial distribution of the radiation intensity is determined with a similar detector.

20. The method according to any one of the preceding claims, characterized in that at least two substances to be analyzed are each marked with a quantity of point radiation objects, the substances marked in this way are mixed and the mixture is then applied to the carrier surface, in particular in the form of a flat analysis means.

21. The method according to claim 20, characterized in that the substances to be analyzed are peptides, proteins and / or oligonucleotides.

22. The method according to claim 20 or 21, characterized in that the analysis means is a protein gel, nucleic acid array, protein array, ELISA array and / or a blot.