Method of combined immunologic and genetic cell investigation

FIELD: medicine.

SUBSTANCE: cell suspension under investigation is incubated with biochip, containing immobilised on biochip antibodies, which have specificity to superficial antigens of investigated cells. After incubation, biochip is washed from non-specifically bound cells. Cells, which remain bound with biochip, are subjected to processing with labelled polynucleotide probes of one or several types with further hybridisation. Reading and processing of results are performed by presence of cell binding in area of biochip sites, containing immobilised antibodies, presence in them of determined superficial antigens is detected, and presence and character of binding of labelled polynucleotide probes are used to determine genetic signs in the same cells.

EFFECT: method application makes it possible to increase quantity of simultaneously determined superficial antigens on different cells with application of non-conjugated with label antibodies, simultaneously reducing number of used antibodies.

9 cl, 2 dwg, 2 ex

 

The invention relates to medicine, in particular to the field of Hematology, Oncology and clinical laboratory diagnostics, in addition, it can be used in veterinary medicine and biology.

There is a method of detection of surface antigens of cells with microchips (see Liu, A.Y. Differential Expression of Cell Surface Molecules in Prostate Cancer Cells// Cancer Research. 2000., vol.60, p.3429-3434), taken as a counterpart, namely, that the biochip (representing a substrate with immobilized in strictly defined areas of antibodies, each specific to a particular cell surface antigen) is incubated with the cell suspension. Cells, having on its surface corresponding antigens bind to the antibodies immobilized in strictly defined areas (spots) of the biochip. The remaining cells are not specifically associated with its surface. Then, the chip is rinsed from the non-specifically bound peroxidase cells isotonic wash solution. As a result, on the surface of the biochip only those cells that are contacted with the antibody. Carry out reading of the defining any spots of the biochip happened binding cells. The method allows simultaneous determination of different cells a large number of different surface antigens.

The disadvantage of this method is impossible is very useful for conducting genetic studies of the same cells, that reduces the information content of the analysis.

A known method for performing the combined immunological and genetic studies of cells, called M-FICTION analysis (multicolor fluorescence immunophenotyping and interphase cytogenetics as a tool for the investigation of neoplasms), taken as a prototype (see, for example, J.I. Martin-controlled by subero, I. Chudoba, L. Harder, S. Gesk, W. Grote, F.J. Novo, M. J. Calasanz, and R. Siebert "Multicolor-FICTION Expanding the Possibilities of Combined Morphologic, Immunophenotypic, and Genetic Single Cell Analyses" / Am J Pathol. 2002 August; 161(2): 413-420.) This method is that cells located on the surface of the slide) is treated with one or more fluorescently-labeled antibodies specific to antigens, washed, fixed, dehydration (with solutions of ethanol with increasing concentrations) and dried. Then the same cells treated with one or more fluorescently-labeled DNA probes complementary to known nucleotide sequences. Perform denaturation of DNA and hybridization, in which each labeled probe binds to complementary him a segment of a gene. Then do the washing. Conducting a study of the drug using a fluorescent microscope with sets of filters, allowing to determine the fluorescence of each of the labels used, and, if necessary, to determine the mutual position of labels. (Cadavid antibodies and each type of probes probe anywhereman with a certain tag). Can be also obtained three-dimensional image of each cell. For receiving a sequence of images at different distances of the lens of the microscope from the drug (for example, with a step of 1 μm) and the exercise of their computer processing.

Performing such analysis allows simultaneous detection of morphologic, immunophenotypic, and genetic features of the same cells.

The disadvantage of this method is the high consumption of expensive fluorescently - labeled antibodies in the determination of the antigens of cells with surface localization. While it is impossible to conduct studies using unlabeled antibodies with lower cost. In addition, due to the limited number of simultaneously used fluorescent labels on different cells can be identified only a small number of surface antigens, which limits the number of simultaneous defined immune cell subpopulations. (In practice, the number of simultaneously used fluorescent labels are limited due to the overlapping spectra of the issue).

Objective of the claimed invention is to reduce the consumption of antibodies in the determination of surface antigens of the cells and increase the number of simultaneously defined surface antigens on different cells, and prob is a possibility of the use of this antibody is not conjugated to a label.

The problem is solved due to the fact that according to the method of combined immunological and genetic studies of cells, namely, that carry out the determination of the antigens of the cells, using antibodies with known specificity, these cells capture and hold hybridization with a labeled polynucleotide probes of one or more species, perform washing, establish the presence or absence of the data cells of one or more desired genetic characteristics of the investigated cell suspension incubated with the chip containing immobilized antibodies specific to surface antigens of the cells are shaded biochip from nonspecific bound peroxidase cells remaining bound cells treated labelled polynucleotide probes one or more species, carry out the hybridization, perform the reading and processing of the result.

Carry out the determination of the co-expression of antigens, including the processing of contacting biochip cells labeled with antibodies specific to one or more antigens.

For studies of the same sample of biological material using a complex of several biochips, contacting which cells are subjected to hybridization with different types of labeled polynucleotide probes or srabatyvayut different types of labeled antibodies.

Determine the density of binding cells in the parts of the biochip with different immobilized antibodies and assess the content in the sample of cells with the appropriate surface antigens.

Determine the percentage of cells simultaneously with defined genetic trait and defined antigen, the number of cells with defined antigen.

Determine the number of cells that have defined coexpression antigens and assess the proportion of cells simultaneously with defined genetic trait.

The use of the claimed invention allows for different cells using the biochip to determine a set of surface antigens. (The number of surface antigens, determined by the fact of linking cells in different test areas of the biochip with immobilized antibodies is determined only by the number of these sites on the substrate of the biochip and can reach several tens or hundreds.). The use of the biochip can significantly reduce the consumption of antibodies.

You can still define multiple antigens on each individual cell using the additional processing of the labeled antibodies. But due to the binding of any one of the cells immobilized on the biochip antibodies on its surface is defined by one ntigen more. While immobilized on the biochip cheaper antibody is not conjugated with a label and consumption of these antibodies is very low.

Use for the analysis of the same sample of biological material several biochips, which are the definition of different types of co-expression of antigens using different types of labeled antibodies or definition is different genetic characteristics using different types of labeled polynucleotide probes increases the informativeness of the study and extends the range of characteristics defined in relation to each other.

Possible qualitative semi-quantitative or quantitative determination of the density of binding cells (ratio of the number of cells to the area of the plot on which they are associated) in the parts of the biochip containing immobilized antibodies. Based on the value of the density of binding may be determined by the content in the sample of cells with defined using the biochip surface antigens.

Can be determined the proportion of cells with coexpression antigens, as well as the percentage of cells with defined genetic characteristics from the number of cells bound peroxidase in the study area of any test section of the biochip with the immobilized antibodies. This allows you to evaluate the content in the sample cell, simultaneously with l the battle of defined (using biochip) surface antigens and coexpression with additionally defined antigen (one or more) and / or one or more defined genetic characteristics. This approach significantly increases the information content of the analysis.

The claimed method can be used, for example, to determine the immunological subpopulations of tumor cells that have a particular genetic (cytogenetic) anomaly that will improve the accuracy of diagnostic tests and avoid some of the controversies in the diagnosis. This method can also be used for many other tasks, in particular, for conducting proteomic studies.

The claimed method is illustrated by illustrations, where

figure 1 presents a diagram showing the result of the study of cells of the patient I., 75 years. The diagnosis of chronic b-cell lymphocytic leukemia (b-CLL).

Figure 2 presents a diagram showing the result of the study of cells of the patient P., 59 years old. Diagnosis: lymphoma cells of the mantle zone. Likemyself.

The claimed method is as follows.

Prior to analysis in a ditch or on the platform (patent for useful model RU 90213) fix biochip, representing a transparent plate, which in strictly defined test areas (spots) of immobilized antibodies specific to surface antigens of the cells. (An example description of a biochip of this class, see: Avestin, Mir is in, Sagaseta, Nguoilon, But, Piitulainen, Aiguablava "Immunological biochips for parallel detection of surface antigens and morphological analysis of cells, Biological membranes, 2008, volume 25, No. 4, s-284). Carry out blocking of nonspecific binding sites of the substrate, if this was not done at the stage of fabrication of the biochip. For this purpose, the chip is incubated with the protein solution (e.g. a solution of nonfat dry milk or bovine serum albumin), and then perform the cleaning solution of detergent (for example, 0.05% solution of the detergent Tween-20 in phosphate buffer). Then carry out incubation biochip with a given cell suspension. The cells are deposited on the surface of the biochip and come into contact with the immobilized antibodies. If the cells have the appropriate surface antigens, they are binding in the test sites (spots) on the surface of the biochip.

Then carry out washing of the biochip to eliminate cells, nonspecific contacting its substrate. For this biochip rinsed several times with isotonic buffer solution. After washing the stained area of the biochip remain bound only those cells that have the appropriate surface antigens. The quality control of washing is performed with p the power microscopic examination. The washing is completed, if the outside spots of the biochip (background areas) associated cells are absent. After washing can be performed by incubation of the biochip with the medium RPMI-1640, which in many cases leads to a significant increase in the bonding strength of the remaining cells with the substrate.

Then carry out the fixation of biochips, for example, a mixture of ethanol and acetic acid (3:1) for 10 minutes in 1% solution of paraformaldehyde for 1 minute. Instead, you may be committed 4% solution of paraformaldehyde or processing carried out in other substances.

Can be processed with a solution of protease in an acidic medium for 5 minutes, followed twice by PBS washing. Then conduct a de-hydration by sequential processing of ethanol with increasing concentration (70%, 85%, and 96% or absolute) for 2 min at +4°C, after which the biochip is dried in the air.

Then, on the surface of the biochip is placed a drop of solutions labeled DNA probes, and served its cover glass. If the chip was mounted in a platform (patent for useful model RU 90213), its removable side with this remove. Carry out incubation at +70°C to +78°C (optimum temperature value for a specific set of probes set experimentally) for 10-15 minutes, and then maintain the ri +37°C for 8-12 hours. This procedure is performed at 100% humidity. Perform washing biochip diluted with citrate buffer (SSC). Under a layer of buffer to gently remove the cover glass. Then biochip process in SSC buffer with the addition of the reagent PN-40 (0,4x SSC/0.3% of the PN-40) for 2 minutes at +73 2 minutes (2x SSC/0.1% of the PN-40) at room temperature and dried. Can be made more staining DNA with a solution of 4',6-diamine-2-phenylindole (DAPI).

On the biochip put a drop of so-called antifade (anti-fade) solution that reduces the "fading" of labels occurring under the action of ultraviolet radiation. Top take cover glass.

After all the manipulations biochip examined using a fluorescent microscope. You get a micrograph of each spot in normal lighting, as well as in ultraviolet light with the use of sets of filters.

Then handle the result. On the microphotographs spots of the biochip, performed at low magnification microscope in normal lighting, the density of binding of cells (number of cells, who contacted per unit area of the surface of the substrate of the biochip).

On the basis of the density values of binding cells in the stained area of the biochip may be determined contents in cell suspension having suitable the antigens. To do this, determine the ratio of the density of binding cells in a specific test area of the biochip to the maximum possible density of binding cells (determined by calculation) or to the density of binding cells in the control plot, providing linking all types of investigated cells present in the sample. (For example, in the study of cells as a control plot of the biochip can be used test area (spot) with immobilized antibodies specific to the antigen CD45, which is present on the surface of all types of leukocytes). The values obtained, expressed in percent, numerically coincide with the values of the percentage of the sample of cells with the appropriate surface antigens. This match is performed, if the incubation of the biochip with the cellular suspension is performed without stirring.

On the microphotographs of multiple fields of view, performed at high magnification microscope with ultraviolet light (using the appropriate sets of filters) in each spot biochip detect the presence of cells with the appropriate label (combination of multiple labels). Determine the ratio of the number of such cells to the total number of cells in the data fields. On this basis, calculate the content of cells with appropriate ejstvujuschij surface antigen (defined using the biochip), and having determined using in citu hybridization genetic trait.

The reading process and result processing can be automated.

When performing genetic studies of contacting biochip cells can also be used polynucleotide probes conjugated (labeled) not with a fluorescent label, and Biotin (or Avidya), or enzyme, for example, peroxidase, or any other substance (see "clinical Molecular diagnostics. Methods"./ Edited Sherington and Gamache, M.: Mir, 1999). In this case, use other protocols in situ hybridization and visualization. The difference will be that these protocols will not be processed smears or fingerprints, and biochips with related cells.

You can use radioisotopic labels. But this approach is not acceptable for most laboratories because of the need to use increased security measures.

With the appropriate hardware and software it is possible to obtain three-dimensional images of contacting biochip cells. For this purpose it is necessary to obtain a series of images of cells bound peroxidase in the region of each of the spots of the biochip, lens-shift microscope with a certain step and to perform computer processing the image is of.

In fact binding in a particular spot biochip each individual cell is determined by only one antigen, but in different cells is determined by the number of surface antigens (equal to the number of kinds of antibodies immobilized on the biochip). If you want each individual cell to determine the presence of more than one antigen at a stage prior to hybridization with polynucleotide probes, process biochip-labeled antibodies specific to one or more antigens (in the latter case, the use of antibodies conjugated with different labels). Before this biochip 10 minutes, fixed in acetone and dried in air. Fixation with acetone is carried out only if it does not dissolve the substrate of the biochip. Can be done recording by treatment with a solution of paraformaldehyde in buffer or fixation using other reagents. In some cases, you can research without committing at this stage. Then within 30 minutes exercise incubation with the appropriate antibody solution (one or more) and spend washing. In addition to the direct immunocytochemical studies can be performed the study one of the indirect methods to achieve significantly greater sensitivity. (See, example is, "Molecular clinical diagnostics. Methods". Edited Sherington and Gamache, M.: Mir, 1999). You need to use the same method for the visualization of the results determine the co-expression of antigens and results of in situ hybridization with polynucleotide probes (for example, and in each case was used fluorochrome label).

If the definition of multiple polynucleotide sequences using probes (labeled with different labels) or additional definition of co-expression of multiple antigens using several types of labeled antibodies (labeled with different labels) for one reason or another cannot be performed on cells that are associated on a single chip, using a complex of several biochips.

Examples of use of the invention.

Example 1.

Using two biochips were investigated tumor cells isolated from blood of a patient I., 75 years old, suffering from chronic b-cell lymphocytic leukemia (b-CLL). Each chip contained test plots with immobilized antibodies specific to antigens CD2, CD3, CD4, CD5, CD7, CD8, CD10, CD16, CD19, CD20, CD22, CD23, CD56, CD44, CD45. Before analysis, the biochips were fixed in cuvettes. The allocation of the leukocyte fraction of the cells was carried out by centrifugation in a density gradient (solution ficoll and urografin). The cell which was reasponsible in phosphate buffer (PBS) supplemented with V / V heat inactivated human serum (15% by volume).

Then within 30 minutes was carried out by incubation of biochips with cell suspension, after which he was made their money from not contacting antibodies cells. For this purpose, the biochips were rinsed with isotonic NaCl, buffered PBS. The quality control of washing was performed using an inverted microscope. After that, the biochips were incubated for 15 minutes in medium RPMI-1640 and washed three times with 0.9% NaCl solution buffered PBS.

Then performed the fixation of biochips in 4% solution of paraformaldehyde for 10 minutes and washed twice by PBS. Then within 5 minutes was treated with a solution of protease in an acidic medium and washed twice by PBS. After that were de-hydration by sequential processing of ethanol with increasing concentration (70%, 85%, and absolute). Then biochips drying in the air.

Next, on the surface of biochips inflicted solutions (samples)containing labeled DNA probes. The surface of biochips were covered with cover glasses. The biochip No. 1 was applied to the samples for the identification of loci on chromosomes R and 11q (labeled with fluorochromes, giving the orange and green luminescence, respectively). On the biochip No. 2 was applied to the samples for the identification of loci: 1) R (labeled fluorochroman giving green glow), 2) 13q14 (labeled fluorochromes, giving an orange glow) 3) 13q34 (labeled fluorochromes, d is the missing green-blue glow). Used reagents manufactured by "Abott-Vysis".

Was carried out by incubation at +78°C for 10 minutes and then kept at +37°C over night. Then biochips for 2 minutes at +73 was treated with a solution containing SSC buffer with the addition of the reagent PN-40 (100 ml 2xSSC 20 ml water, 80 ml, PN-40 300 μl). Under the buffer layer were carefully removed the glass cover. Then biochips for 2 minutes at room temperature was treated with a solution containing SSC buffer with the addition of the reagent PN-40 (100 ml 2xSSC to 99.9 ml, PN-40 100 μl) and dried. On each biochip has caused a drop antifade solution. On top put a cover glass.

After all the manipulations biochips examined using a fluorescent microscope. When this was received micrograph of each spot in normal lighting, as well as in ultraviolet light with the use of sets of filters.

Did the photographs of the test sites biochip under normal and ultraviolet light. On the microphotographs made in the ordinary illumination at low magnification (lens magnification microscope X10) determined the density of binding of cells (number of cells, who contacted the plot of a given area) in the region of each test section of the biochip. On this basis, the estimated content in the sample cell, and ausich each of defined antigens. On the microphotographs of test plots biochip made in ultraviolet light at high magnification (lens h) in different fields of view, was determined by the presence of cells with desired diagnostically meaningful combination of fluorescent labels.

The patient was discovered trisomy of chromosome 12. This was manifested in the fact that some of the cells associated with the biochip No. 2 had 3 signal (fluorescent area) green. Other genetic disorders (diagnostically significant combination of tags), as defined by data sets of probes (tick R, 11q, and 13q14 and 13q34), were not found.

Was determined by the ratio of the number of cells with the characteristic of trisomy 12 chromosomes to the total number of cells in these microphotographs spots of the biochip. On this basis, the content was determined in the sample of cells with the appropriate sign.

The results obtained are given in figure 1

Example 2.

Using biochip containing the same set of immobilized antibodies were investigated cells of the patient P., 59 years old, with a diagnosis of lymphoma cells in the mantle zone. The patient was observed likemyself. The study was carried out similarly as described in example 1. Used test to determine the translocation t(11;14)(q13;q32). The probe communicates with the gene CCND1 was anywhereman the nous, with orange fluorescence and probe binding to the IgH gene was anywhereman labeled with green fluorescence.

A significant part of the investigated cells of this patient was identified this translocation. In cells that do not have this translocation was determined in 4 separate signal: 2 orange and 2 green. In cells with the translocation was observed 2 yellow signal (continuous) and 2 separate signal 1 green and 1 orange. In the same way as in example 1, it was found the contents of cells expressing each of which is defined by the biochip antigens and still having this characteristic. The results obtained are given in figure 2.

1. The method combined immunological and genetic studies of cells, wherein the cell suspension is incubated with the chip containing immobilized antibodies specific to surface antigens of the cells, after incubation biochip washed from nonspecific bound peroxidase cells, then the chip is subjected to the processing of labeled polynucleotide probes of one or more species, carry out the hybridization, read, and process the received results, and establish the presence of the investigated cells defined surface antigens, and the presence or absence of the same cells defined genetic the definition of signs.

2. The method according to claim 1, wherein after performing the washing of the biochip from the nonspecific bound peroxidase cells remaining bound to the biochip cells subjected to fixing, de-hydration and drying.

3. The method according to claim 1, characterized in that after performing hybridization with a labeled polynucleotide probes are shaded biochip from not bound peroxidase polynucleotide probes.

4. The method according to any one of claims 1 and 3, characterized in that after performing the washing of the biochip from unbound polynucleotide probes spend it drying.

5. The method according to claim 1, characterized in that the processing results determine the density of binding cells in the parts of the biochip with different immobilized antibodies and evaluate the content in the sample of cells with the appropriate surface antigens.

6. The method according to any one of claims 1 to 5, characterized in that to determine the proportion of cells sachetana with defined genetic trait and defined antigen, the number of cells with defined antigen.

7. The method according to claim 1, characterized in that to determine the co-expression of antigens contacting the biochip further treated cells labeled with antibodies specific to one or more antigens.

8. The method according to any one of claims 1 and 7, wherein the examining of the same cell suspension using a complex of several biochips contacting which cells are subjected to hybridization with different types of labeled polynucleotide probes or process different types of labeled antibodies.

9. The method according to any one of claims 1 and 7, characterized in that to determine the number of cells that have defined coexpression antigens and assess the proportion of cells simultaneously with defined genetic basis.



 

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2 tbl, 2 ex, 4 cl

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10 dwg, 2 tbl, 4 ex

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1 tbl, 3 ex

FIELD: medicine.

SUBSTANCE: what is offered is a method of marking a human 7th chromosome providing in situ hybridisation of metaphasic or interphasic cell chromosomes of a tested sample and a DNA probe presented by a marker plasmid alpha R1-13 which consists of EcoR 1-EcoR l DNA fragment of a pBR 325 vector of the value 5966 base pairs and EcoRl-EcoRl alphoid DNA fragment of the human 7th chromosome of the value 680 base pairs. The testing environment in which the used DNA-probe specifically interacts with a centromeric region of the 7th chromosome without cross hybridisation with other human chromosomes are developed.

EFFECT: more efficient identification of the presented chromosome and enabled application of the new method in medical diagnostics.

4 ex

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