Method for identifying cell specific target structures

The invention relates to a method for identifying cell-specific target structures.

The identification of cell-specific target structures is of central importance in the Elucidation of cell-cell interactions, the countless effects within a can drag organism itself. In particular, the disease-specific knowledge Targets is a crucial prerequisite for the development of effective and simultaneously less adverse effects drugs.

It is known that immune cells (lymphocytes), specific combinations of proteins, also protein combination patterns or briefly called PKM, express that a bond are responsible to endothelial cells of the blood vessels of the brain and muscle tissue. Other Combinations of proteins not lead other hand capable of binding to these Endothelial cells. Surprisingly, these specific combinations are interindividually constant and have always the same binding functions. It appears therefore at the specific protein combination patterns to a constant interindividual Lymphocyte binding code of cell surface for organ-specific endothelial cell surface to act, which is a cell-specific target structure. cell-specific Target structures may thus have very specific protein combination patterns.

Invasive tumor cells have specific protein combination patterns on their Cell surface, leading to a targeted, ie organ-selective invasive behavior. Such protein combination patterns therefore provide targets for potential drugs represent.

An absolute condition for the development of such highly selective drugs However, the knowledge of the molecular composition of these target structures.

A method for identifying target structures are known from the prior art, in which on the analysis of gene expression profiles of diseased tissues or cells Compared to gene expression profiles of healthy tissues or cells are based, said both protein expression profiles and expression profiles of messenger ribonucleic acid (MRNA) light on the appearance of new proteins, or dysregulated to give abnormally modified proteins in diseased tissues or cells (eg, in: F. Lottspeich / H.Zorbas; bioanalysis; Oxford University Press; Heidelberg, 1998).

However, these methods all go from cell homogenates from, which usually thousands or millions of cells are based, since only by means of these cell volumes can be created expression profiles of the abovementioned type. In the cell homogenates are the cells in more open form, so that the proteins or mRNA molecules with can help biochemical methods extracted and separated.

A disadvantage of these known methods, however, is that they are not capable, identify protein combination patterns, since the individual protein components of a such protein combination pattern by the generation of cell homogenates and by the subsequent extraction operations are completely separated and the essential lost information with respect to their cellular and gewebetopologischen location goes. By Destruction of the cell compartments can continue any information regarding the Combinations of proteins within these cell compartments and their relative topological relation obtained another.

Another disadvantage of the known method is, moreover, that no analysis on the is the single cell level feasible, so that differences between the individual cells with respect to their Protein combination patterns are undetectable. In addition, proteins that can only small amount present, will not be detected by the known methods. This relates in particular proteins or specific protein combinations, for example, only in a few, but pathogenic, disease-specific cells vorkommmen.

Another disadvantage of the known method is that the preparation steps of Tissue or cells of their removal or extraction to the step of isolating or separation of proteins of a large number of variable external influences may be subject, which are difficult to control and unify.

The object of the invention is therefore to provide a method of the above type, can be identified by the cell-specific protein combination patterns and the mentioned drawbacks of the prior art are overcome.

This object is achieved by an inventive method for the identification of cell-specific target structures comprising the steps of: (a) automated application of a reagent solution Y1 having at least one marker molecule, to an object X1 the cells and / or cell membranes comprising the stem from a cell or tissue sample; (B) exposing the reagent solution Y1 and automated detection of at least one Marking pattern of the labeled with the reagent solution Y1 object X1; (C) removing of the reagent solution Y1 before or after detecting the marking pattern, and Repeating steps a) and b) with further reagent solutions Yn (n = 2, 3 ... N) each of the at least one marker molecule and / or at least one other exhibit marker molecule; detected (d) Summarize each in step b) Marking pattern to a complex molecular combination pattern of the object X1; (E) repeating steps a) to d) with at least one further object Xn (n = 2, 3 ... N), while the other cells and / or other cell membranes having the cell of another or tissue sample taken from; (F) determining at least one difference between the Combination pattern of the object X1 and the combination pattern of the object Xn; (G) Identifying at least one reagent solution Y1 or Yn whose marking pattern to in step f) obtained at least causes a difference; and (h) selecting Molecules or molecular complexes, which through the at least one marker molecule in the Step g) identified reagent solution Y1 or Yn are bound, from a Homogenate of cells and / or cell membranes of the cell or tissue sample to the differing according to step f) object Xn originate; and (i) biochemical Characterizing the selected according to step h) molecules or molecular complexes.

The inventive process the protein combination patterns can single Cells or cell membranes of different cell or tissue samples comparative to be examined. In this case, those Markiermoleküle can be identified, the for example, to a specific protein combination patterns or to a particular binding region of such a protein combination pattern of a first object, the one first tissue or cell sample is taken, and at the same time not to a second object tie that comes from a second tissue or cell sample. Using these identified Markiermoleküle can now using a sample portion of the first tissue and / or cell sample molecular those regions (molecules or molecular complexes) of discovered protein combination pattern or selected and subsequently characterized are to be bound by the identified Markiermoleküle. is In this way, it possible to have the molecular composition of a protein combination pattern, the Arrangement of the molecules within the protein combination pattern and arrangement of Protein combination pattern to detect within a tissue or a cell.

A particularly advantageous development of the invention provides that the h in step) used homogenate prior to step h) by molecular or molecular complex separation processes, especially Protei ntrennverfahren, is separated into individual Homogenatbestandteile. In this way, the selection of the molecules or molecule complexes of the Homogenate greatly simplified.

A further advantageous development of the method provides that the Object X1 cells and / or cell membranes having the one of a cell or tissue sample ill patients originate, and in that at least one other object Xn cells and / or comprises cell membranes of a cell or tissue sample of a healthy subject come. Thus, disease-specific targets and the corresponding Protein combination patterns are identified, so that this basis of knowledge developed disease-specific protein combination patterns highly specific drugs may be, which are almost free of side effects due to this very specificity.

A further advantageous development of the invention provides that the method comprises parallel step of: (x) creating a respective protein expression profile of each a sample portion of the cell or tissue samples, from which cells and / or cell membranes enter into the objects X1 and Xn, and comparing the allocated object X1 Protein expression profile with the assigned object Xn protein expression profile, wherein at least one difference is detected.

Furthermore, the inventive method can comprise, after step x) the following step: (Y) Make at least one protein and / or at least one protein modification, the or the causes identified in step x), except with respect to a bond of at least one Markiermoleküls identified in Step g) reagent solution Y1 or Yn. In this way it can be determined whether it is the case by comparing the Protein expression profiles observed differences to procedural artifacts (see above) or to actually significant differences are, as they also by the The method according to the invention could be determined. The inventive method can thus be used as an essential complement to the methods already known will.

In another particularly advantageous embodiment of the invention it is provided that at least one marker molecule used in step a) and / or step c) at least one Protein and / or at least one protein modification binds the one or more in step x) caused ascertained difference. can by known methods, for example, Antibodies or ligands are developed, the proteins or protein modifications bind that of by comparing protein expression profiles as areas Targets were identified. Through the use of these antibodies or ligand in the The method according to the invention can be checked whether the comparison of by the produce protein expression profiles identified proteins or protein combination patterns not.

Furthermore, at least one, in step a) and / or step c) marker molecule used be fluorochrome conjugated. Such a fluorescent label is a marker molecule particularly easily detectable.

Furthermore, at least one, in step a) and / or step c) marker molecule used be an antibody, wherein the antibody is an antibody library be found in can, the antibody library is a naive or a non-naive antibody library can be. In a so-called naive antibody library is a Library whose antibodies have no known specificity, while the antibodies the non-naive antibody library known molecules, such as proteins or glycoproteins recognize. By using a non-naive library therefore is the same for identification an antibody to an information available, which molecule or molecules which the antibody binds.

Furthermore, at least one, in step a) and / or step c) marker molecule used be a ligand. The ligand may be taken from a ligand library, the Ligand library may be a naive or a non-naive ligand library. The Designations "naive" and "non-naive" are analogous to the above statements relating to understand.

After removing the reagent solution according to step c) may be followed by a washing step, in the applied wash solution to the object X1 and after a predetermined time is removed. In this way excluded that a newly applied Reagent solution is contaminated by a previously applied reagent.

Step d) can be carried out advantageously by computerized image overlays.

The process can be repeated as often as desired bleaching cycles, in particular after step b) include. Such bleaching cycles prevents marks which already have been detected, can be detected again in a subsequent step.

Further details, features and advantages of the invention will become apparent from the following Description of an embodiment.

In the embodiment, a tissue or cell sample is divided into two samples of play divided. The first sample portion is used as a starting material for the manufacture of Homogenate for biochemical protein characterization by the known processes. In particular, methods such as 2D gel electrophoresis, for the creation of Protein expression profiles applied.

The second sample portion is used to produce tissue sections or cell preparations from with intact cells. If it is in the second sample portion to a Tissue block, as is established from this, a tissue section. If it is however to Cells are in a suspension, these cells in intact form to a surface in particular applied to a microscope slide. The first object obtained in this manner is subjected to the following automated method steps:<sl><li>1. receiving a first reagent solution Y1 having one or more fluorochrome Antibodies of a non-naive antibody library;</li><li>2. pipetting the reagent solution Y1 to the first object;</li><li>3. Incubation of the object with this solution Y1 at a specific temperature, especially room temperature;</li><li>4. Remove the reagent;</li><li>5. One-time or repeatedly dropping a washing solution, and then Remove;</li><li>6. applying a buffer solution to the first object;</li><li>7. Detection of the fluorescence distribution pattern, wherein when using several used fluorochromes selective fluorescence recording filters during image acquisition will. In this step, it is determined whether and at which locations of the antibody or show in the sample bond signals. There are positive signals and negative signals equally, for each pixel or location point of the cells or tissue of the first Object digitally recorded;</li><li>8. pipetting a further wash solution;</li><li>9. bleaching of the sample using a fluorescence excitation, wherein the bleaching process is terminated when no fluorescence can be detected;</li><li>10. Remove the further wash solution;</li><li>11. Recording of a reagent solution Y2 with one or more fluorochrome also Antibodies of another or the same antibody library;</li><li>12. pipetting of the reagent solution Y2 on the first object.</li></sl>

This process is continued by repeating the steps 1 to 10, wherein the Reagent Y3, Y4, Y5..Yn be used, the antibodies or ligands of may contain non-naive or naive libraries. One and the same object can on Thus, a completely naive library or with a completely non-naive be studied library, or with a mixed non-naive and naive library.

Each of these process cycles (steps 1 through 10) by registering a corresponding phase contrast image or differential interference contrast image ended.

From the registered respectively in step 7 marking patterns are the existing Signal combinations and the non-existing signal combinations holistically a combination pattern shown determined by computerized image overlays.

This established for the first object combination pattern with other Combination patterns of different samples or subjects cells or conditions or Diseases compared, standardized by the same reagent solutions according to the same above process steps were analyzed.

If this comparison unique, examined for the sample or for a state, a cell shape, a disease or a function-specific signal combinations result, these signal combinations are based on specific, ie selective molecular Interactions and therefore characterize the state of the disease, the function or Cell shape. The detected signal combinations, the corresponding antibodies or Ligands are associated.

The filtered in this way antibodies and / or ligands are now to used in the first sample portion to those proteins, glycoproteins or other Carbohydrate structures "fish out" that specifically, these ligands or antibodies can bind. Given the well-known in the biochemical analysis methods used. The molecules thus found can choose from single molecular species or complexes be different molecular species. In both cases, they provide highly specific Targets, in particular for the development of drugs represent.